**Sceletium Plant Species: Alkaloidal Components, Chemistry and Ethnopharmacology**

Srinivas Patnala and Isadore Kanfer

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66482

#### **Abstract**

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html#ixzz4TlfGGpLw [Accessed: January 12, 2017]

The genus *Sceletium,* classified under the Aizoaceae family, is indigenous to the Western, Eastern and Northern Cape province of South Africa. There are currently eight reported species divided into two main "types" with five species in the *tortuosum* and three in the *emarcidum* type. It has been observed that, in general, mesembrine‐type alkaloids such as mesembrenol, Δ<sup>7</sup> mesembrenone, mesembranol, mesembrenone, mesembrine and epime‐ sembranol as well as some non‐mesembrine type such as Sceletium A4, tortuosamine and joubertiamine occur in the *tortuosum* type; the *emarcidum* type is devoid of alkaloids. Morphological identification of species type presents a formidable challenge, where subtle differences are found in the secondary veins that branch off from the middle vein toward the leaf margin. In view of the fact that the plant contains a complex mixture of closely related compounds, in particular alkaloidal components, separation techniques and their application to evaluate specific chemical components are an important aspect which permits accurate characterization and quantification. In addition, the develop‐ ment of appropriate analytical methods for chemotaxonomic studies has provided valu‐ able information to confirm specific plant identity. Importantly, these methods are also required for the quality control of plant material used to manufacture complementary and traditional medicines containing *Sceletium*.

**Keywords:** *Sceletium tortuosum*, *Sceletium emarcidum*, alkaloids, mesembrine, chemotaxonomy, HPLC‐MS

#### **1. Introduction and background**

Sceletium alkaloids have been studied over a century when their presence was first reported in1896 and later by Zwicky in 1914. In a detailed study by Zwicky on about 40 species of the genus *Mesembryanthemum,* more than 50% of the plants tested positive for alkaloids. Due to

this large number of species, the genus *Mesembryanthemum* was abandoned and some of the species were reassigned to genus *Sceletium*, family Aizoaceae [1].

These alkaloids, originating from *Sceletium* plants species, were widely found in the Western and Karoo regions of South Africa. The name *Sceletium* is derived from the Latin word *Sceletus* meaning skeleton. The derivation of the name is due to the prominent lignified leaf vein structure that is observed in dried leaves of this genus which give a skeletal appearance. Anecdotal evidence suggests that this plant is highly revered and held in great esteem by the tribes who collected and bartered it frequently in exchange for cattle and other commodities. Subsequently, the early Dutch colonists further showed commercial interest in this plant, and many plants of this family were also introduced to European cultivation [2].

The *Sceletium* plants can readily be identified by its persistent dry "skeletonized" leaves which enclose the young leaves during the dry season (**Figure 1a**), to protect them from adverse environmental conditions [3]. The specimens of two main types of Sceletium plants: *Sceletium tortuosum* and *Sceletium emarcidum* are depicted in **Figure 1b** and **c**, respectively.

**Figure 1.** (a) Skeletonized leaves of *S. tortuosum*. (b) *Sceletium tortuosum*. (c) *Sceletium emarcidum* (with skeletonized leaves).

#### **2.** *Sceletium* **species**

Sceletium species occurs in the Eastern, Northern, Western Cape provinces of South Africa and the genus *Sceletium,* belongs to the family, Aizoaceae [1].

#### **2.1. Identification of** *Sceletium* **plant species**

The specimens were studied and identified using the identification key of Gerbaulet [1]. Based on the identification key, the venation pattern which differs between species is one of the important taxonomic identification features.

There are currently eight reported species [3] of this genus, divided into two "types" with five species in the *tortuosum* type and three in *emarcidum* type as follows:

*Tortuosum* type: *Sceletium tortuosum; Sceletium crassicaule; Sceletium strictum; Sceletium expan‐ sum and Sceletium varians.*

*Emarcidum* type*: Sceletium emarcidum; Sceletium exalatum and Sceletium rigidum.*

The main differences are found in the secondary veins that branch off from the middle vein toward the leaf margin. Based on the venation type, the species is mainly classified as either *emarcidum* or *tortuosum* types (**Figure 2**). In the *emarcidum* type, the leaf is more flat and the dried leaf venation pattern shows a central main vein with the curved secondary vein which branches off the main vein, reaching the leaf margins.

In plants of the *tortuosum* type (**Figure 2**), the dry leaves are more concave and usually show about three to five or sometimes up to seven major parallel veins. The secondary veins run straight up to the apex on both sides of the middle vein.

**Figure 2.** Venation pattern of skeletonized leaves in *Sceletium* species. mv = middle vein, csv = curved secondary vein, ssv = straight secondary vein.

#### **3. Chemistry of** *Sceletium* **alkaloids**

this large number of species, the genus *Mesembryanthemum* was abandoned and some of the

These alkaloids, originating from *Sceletium* plants species, were widely found in the Western and Karoo regions of South Africa. The name *Sceletium* is derived from the Latin word *Sceletus* meaning skeleton. The derivation of the name is due to the prominent lignified leaf vein structure that is observed in dried leaves of this genus which give a skeletal appearance. Anecdotal evidence suggests that this plant is highly revered and held in great esteem by the tribes who collected and bartered it frequently in exchange for cattle and other commodities. Subsequently, the early Dutch colonists further showed commercial interest in this plant, and

The *Sceletium* plants can readily be identified by its persistent dry "skeletonized" leaves which enclose the young leaves during the dry season (**Figure 1a**), to protect them from adverse environmental conditions [3]. The specimens of two main types of Sceletium plants: *Sceletium* 

Sceletium species occurs in the Eastern, Northern, Western Cape provinces of South Africa

**Figure 1.** (a) Skeletonized leaves of *S. tortuosum*. (b) *Sceletium tortuosum*. (c) *Sceletium emarcidum* (with skeletonized leaves).

The specimens were studied and identified using the identification key of Gerbaulet [1]. Based on the identification key, the venation pattern which differs between species is one of

There are currently eight reported species [3] of this genus, divided into two "types" with five

*Tortuosum* type: *Sceletium tortuosum; Sceletium crassicaule; Sceletium strictum; Sceletium expan‐*

and the genus *Sceletium,* belongs to the family, Aizoaceae [1].

species in the *tortuosum* type and three in *emarcidum* type as follows:

*Emarcidum* type*: Sceletium emarcidum; Sceletium exalatum and Sceletium rigidum.*

**2.1. Identification of** *Sceletium* **plant species**

the important taxonomic identification features.

species were reassigned to genus *Sceletium*, family Aizoaceae [1].

86 Alkaloids – Alternatives in Synthesis, Modification and Application

many plants of this family were also introduced to European cultivation [2].

*tortuosum* and *Sceletium emarcidum* are depicted in **Figure 1b** and **c**, respectively.

**2.** *Sceletium* **species**

*sum and Sceletium varians.*

Preliminary studies on *Sceletium* were done by Meiring in 1896, suggesting that the presence of alkaloids and this was confirmed by Zwicky in 1914. Further studies on *S. expansum* and *S. tortuosum* reported by Zwicky in 1914, yielded a noncrystalline alkaloid which was named "mesembrin" with the reported molecular formula, C16H19NO4 [4]. Rimington and Roets [5] reinvestigated this plant in 1937, and attempts to crystallize the alkaloid as a free base or hydrochloride salt were unsuccessful. In their experiments, they managed to obtain a crystal‐ line picrate and platinichloride from the methylated free base and the molecular formula was deduced based on combustion analysis. The molecular formula for "mesembrin" was reas‐ signed as C17H23NO<sup>3</sup> and is presently known as mesembrine, suggesting that the molecule belonged to the tropane ester alkaloid group.

Bodendorf and Krieger [6], in their work in 1957, revisited the molecule and successfully crystallized the mesembrine base to its hydrochloride salt, along with isolation of two more bases, namely "mesembrinine," presently known as mesembrenone, which has two hydro‐ gen atoms less, and the structure is closely related to mesembrine. The other base was called "channaine," which was described as a phenolic base, and it was also reported that all these three compounds were purported to be optically inactive.

Popelak and Lettenbauer [7] in 1967 reported the incidence of *Sceletium* alkaloids in the plants they studied as 1 to 1.5%, which consisted of approximately 0.7% mesembrine and 0.2% "mesembrinine." The structure of mesembrine, deduced from their study, was reported as *N*‐methyl‐3a‐(3′,4′‐dimethoxyphenyl)‐6‐oxo‐*cis*octahydroindole, which provided the founda‐ tion for continued studies on this group of alkaloids [4].

Jeffs *et al* in 1974 [8] worked further on *S. namaquense* and *S. strictum* and reported five new alkaloids, namely *Sceletium* alkaloid A4, N‐formyltortuosamine, 4′‐O‐demethylmesembre‐ none, ∆<sup>7</sup> mesembrenone and sceletenone. It was also reported that in a concurrent study by Wiechers *et al* on *S. tortuosum*, another base, tortuosamine, was isolated and had a close struc‐ tural relation to *Sceletium* alkaloid A4.

Arndt and Kruger in 1970 [9] reported three new alkaloids, joubertiamine, dihydrojouber‐ tiamine and dehydrojoubertiamine from *S. joubertii*, where their basic skeletons were bioge‐ netically closely related to mesembrane (**Figure 3**) and not related to the mesembrine—like of alkaloids. The above alkaloids were also isolated and reported in another *Sceletium* species, *S. subvelutinum*, by Herbert and Kattah 1990 [10].

Whereas the phytochemical content of *Sceletium* species has been studied since 1896 [4], the reported alkaloidal content has been constrained to tortuosum‐type species only, and related information on the emarcidum species has been conspicuously absent from the literature. However in 2013, Patnala and Kanfer reported the complete absence of mesembrine as well as other alkaloids usually found in the tortuosum type in their investigations involving three emarcidum species: *S. emarcidum, S. exalatum* and *S. rigidum* [11].

The alkaloids which have been isolated from *Sceletium* species are broadly classified into four structural classes. The major subgroup being the 3a‐aryl‐cis‐octahydroindole skeleton which is referred to as the mesembrine group (**Table 1**) which includes ∆4 series and ∆<sup>7</sup> series based on the double bond at position 4–5 (**Table 2**) and 7–7a (**Table 3**), respectively. *Sceletium* alkaloid A4 (**Table 4**) constitutes the lone member of the second subgroup. The third subgroup is closely related to the second, which is the alkaloid, tortuosamine type (**Table 5**), and the fourth group is the joubertiamine type (**Table 6**), which is closely related to the mesembrine series [10].

Of the above subgroups, the mesembrine type is the largest, consisting of about 15 alkaloids. The class derives its name from mesembrine, which was the first structurally characterized alkaloid molecule [4].

The major alkaloid in mesembrine type is (–)‐mesembrine, reported to be present in up to 1% in *S. namaquence* and occurs as a partial racemate in *S. strictum* and *S. tortuosum* in smaller amounts [8]. The reported alkaloids in this subgroup are listed in **Tables 1**–**3** [12].


#### **3.1. Mesembrine‐type (I)**

"channaine," which was described as a phenolic base, and it was also reported that all these

Popelak and Lettenbauer [7] in 1967 reported the incidence of *Sceletium* alkaloids in the plants they studied as 1 to 1.5%, which consisted of approximately 0.7% mesembrine and 0.2% "mesembrinine." The structure of mesembrine, deduced from their study, was reported as *N*‐methyl‐3a‐(3′,4′‐dimethoxyphenyl)‐6‐oxo‐*cis*octahydroindole, which provided the founda‐

Jeffs *et al* in 1974 [8] worked further on *S. namaquense* and *S. strictum* and reported five new alkaloids, namely *Sceletium* alkaloid A4, N‐formyltortuosamine, 4′‐O‐demethylmesembre‐

Wiechers *et al* on *S. tortuosum*, another base, tortuosamine, was isolated and had a close struc‐

Arndt and Kruger in 1970 [9] reported three new alkaloids, joubertiamine, dihydrojouber‐ tiamine and dehydrojoubertiamine from *S. joubertii*, where their basic skeletons were bioge‐ netically closely related to mesembrane (**Figure 3**) and not related to the mesembrine—like of alkaloids. The above alkaloids were also isolated and reported in another *Sceletium* species, *S.* 

Whereas the phytochemical content of *Sceletium* species has been studied since 1896 [4], the reported alkaloidal content has been constrained to tortuosum‐type species only, and related information on the emarcidum species has been conspicuously absent from the literature. However in 2013, Patnala and Kanfer reported the complete absence of mesembrine as well as other alkaloids usually found in the tortuosum type in their investigations involving three

The alkaloids which have been isolated from *Sceletium* species are broadly classified into four structural classes. The major subgroup being the 3a‐aryl‐cis‐octahydroindole skeleton which is

the double bond at position 4–5 (**Table 2**) and 7–7a (**Table 3**), respectively. *Sceletium* alkaloid A4 (**Table 4**) constitutes the lone member of the second subgroup. The third subgroup is closely related to the second, which is the alkaloid, tortuosamine type (**Table 5**), and the fourth group is the joubertiamine type (**Table 6**), which is closely related to the mesembrine series [10].

Of the above subgroups, the mesembrine type is the largest, consisting of about 15 alkaloids. The class derives its name from mesembrine, which was the first structurally characterized

series and ∆<sup>7</sup>

series based on

mesembrenone and sceletenone. It was also reported that in a concurrent study by

three compounds were purported to be optically inactive.

88 Alkaloids – Alternatives in Synthesis, Modification and Application

tion for continued studies on this group of alkaloids [4].

tural relation to *Sceletium* alkaloid A4.

*subvelutinum*, by Herbert and Kattah 1990 [10].

emarcidum species: *S. emarcidum, S. exalatum* and *S. rigidum* [11].

referred to as the mesembrine group (**Table 1**) which includes ∆4

none, ∆<sup>7</sup>

alkaloid molecule [4].

**Figure 3.** Mesembrane.

**Table 1.** Mesembrine‐type (I) *Sceletium* alkaloids.

#### **3.2. Δ⁴ Mesembrine‐type (II)**


**Table 2.** Δ4 Mesembrine‐type (II) *Sceletium* alkaloids.

#### **3.3. Δ⁷ Mesembrine‐type (III)**

**Table 3.** Δ<sup>7</sup> Mesembrine‐type (III) *Sceletium* alkaloid.

#### **3.4.** *Sceletium* **A4 types (IV)**

**Table 4** depicts *Sceletium* A4 alkaloid (16) and is reported to occur in *S. namaquense* as an optically active crystalline base. The other reported alkaloid [8] which is closely related to this structure is a noncrystalline optically active compound mentioned as dihydropyridone base (17).

**Table 4.** *Sceletium* A4 type (IV) alkaloids.

#### **3.5. Tortuosamine type (V)**

The reported alkaloids (**Table 5**) in this subclass are tortuosamine (18), N‐formyltortuosamine (19) and N‐acetyltortuosamine (20). Tortuosamine, a noncrystalline optically active base, was isolated from *S. tortuosum* [8].


**Table 5.** Tortuosamine‐type (V) *Sceletium* alkaloids.

#### **3.6. Joubertiamine types**

These alkaloids are reported to occur principally in *S. joubertii* and have also been reported to occur in *S. subvelutinum*. These alkaloids are further classified as depicted in **Tables 6**–**8** [8].


#### *3.6.1. Dihydrojoubertiamine (VI)*

**3.4.** *Sceletium* **A4 types (IV)**

90 Alkaloids – Alternatives in Synthesis, Modification and Application

**3.5. Tortuosamine type (V)**

**Table 4.** *Sceletium* A4 type (IV) alkaloids.

isolated from *S. tortuosum* [8].

**3.6. Joubertiamine types**

**Table 5.** Tortuosamine‐type (V) *Sceletium* alkaloids.

base (17).

**Table 4** depicts *Sceletium* A4 alkaloid (16) and is reported to occur in *S. namaquense* as an optically active crystalline base. The other reported alkaloid [8] which is closely related to this structure is a noncrystalline optically active compound mentioned as dihydropyridone

> **No. R1 R2 Compound** 16 OMe OMe *Sceletium* A4

17 OMe OMe Dihydropyridone base

The reported alkaloids (**Table 5**) in this subclass are tortuosamine (18), N‐formyltortuosamine (19) and N‐acetyltortuosamine (20). Tortuosamine, a noncrystalline optically active base, was

> **No. R1 R2 R3 Compound** 18 OMe OMe H Tortuosamine

19 OMe OMe CHO N‐formyltortuosamine 20 OMe OMe COMe N‐acetyltortuosamine

These alkaloids are reported to occur principally in *S. joubertii* and have also been reported to occur in *S. subvelutinum*. These alkaloids are further classified as depicted in **Tables 6**–**8** [8].

**Table 6.** Dihydrojoubertiamine‐type (VI) *Sceletium* alkaloids.

#### *3.6.2. Dehydrojoubertiamine (VII)*

**Table 7.** Dehydrojoubertiamine‐type (VII) *Sceletium* alkaloid.

#### *3.6.3. Joubertiamine (VIII)*

**Table 8.** Joubertiamine‐type (VIII) *Sceletium* alkaloids.



**Table 10.** Physicochemical characteristics of some typical non‐mesembrine‐type *Sceletium* alkaloids.

The physicochemical characteristics of various Sceletium alkaloids—mesembrine‐type and non‐mesembrine‐type alkaloids are compiled in **Tables 9** and **10**.

#### **4. Extraction, isolation, synthesis and characterization of** *Sceletium* **alkaloids**

Natural products are known to contain complex chemical components. Hence, it is essential that active components in such products are identified and analyzed by validated methods to ensure product quality. The development and validation of the requisite analytical method and procedures for QC can only be achieved by testing the product using qualified reference substances.

Several methods have been reported for the extraction and isolation of these alkaloids from *Sceletium* species. In 1937, Rimington and Roets [14] described their extraction procedure of *Sceletium* alkaloids, and subsequently in 1957, Bodendorf and Krieger [6] published a dif‐ ferent extraction procedures. Popelak and Lettenbauer, in 1967 [7], reported the isolation of some alkaloid bases along with mesembrine and mesembrinine and prepared their hydro‐ chloride salts. Arndt and Kruger [9] reported an extraction procedure of the aerial parts of *Sceletium joubertii* to obtain those relevant alkaloids.

Herbert and Kattah [10] in their biosynthesis study of alkaloids in *Sceletium subvelutinum* reported the isolation and purification of joubertiamine and related alkaloids. Jeffs et al. [8]

**Structure[21]**

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

HCl

OCH3

N

CH H

3

**Alkaloid**

**MW**

**MF**

**Description**

**OR**[ *α*] 20

‐55.4° (MeOH)

‐8.4° (MeOH)

 racemic

 –

–

–

‐32°(CHCl3), ‐30° (C

‐3.2° (C

H2 OH)

† ‐13°

5

†

H2 OH)

5

144–145°C

[4], †

[7]

[4], †

[7]

[4]

178–185°C

*D*

> **BP**

**MP** **Reference**

[4], \*[13]

[4]

[4], †

[7] MW, Molecular weight; MF, molecular formula; OR, optical rotation; BP, boiling point; MP, melting point; MeOH, methanol.

**Table 9.** Physicochemical characteristics of mesembrine‐type *Sceletium* alkaloids.

[4]

\*186–190°C

 –

205–206°C

†88–89°C

 –

Pale yellow

Needle‐shaped

Pale yellow

Low melting solid

 Pale brown

crystalline

powder

viscous liquid

viscous liquid

crystals

C17H23NO3

C17H23NO3.HCl

 C17H21NO3

C17H21NO3

C17 H23 N O3

C17H25NO3 Cubic crystals

Pale brown oil

 –

C17H25NO3

C16H23NO3

289.36

325.80

 287.36

287.36

291.39

291.39

275.15

(–)‐Mesembrine

HCl

(–)‐Mesembrine

Mesembrenone

Δ7 Mesembrenone

Mesembrenol

(–)‐Mesembranol

Epimesembranol

 (–)‐N‐ Demethylmesembranol

92 Alkaloids – Alternatives in Synthesis, Modification and Application

O

N

CH3

O

N

CH3

O

N

CH3

O

N

OH

OH

N

OH

N

H

OH

H

CH3

H

N

CH3

H

CH3

H reported the extraction of alkaloids from *Sceletium namaquense* which yielded mesembrine, mesembrenone, *Sceletium* A4, N‐formyltortuosamine, ∆<sup>7</sup> mesembrenone, tortuosamine and some unidentified alkaloids. Smith et al. [15] extracted mesembrenol (Table 2, No. 9) {incor‐ rectly designated as 4′‐*O*‐demethylmesembrenol and labeled (1) in their paper}, mesembrine and mesembrenone from Sceletium plant material. Gericke et al. [16] in their US patent appli‐ cation described the extraction of mesembrine‐type alkaloids with a yield of between 15 and 35 mg per gram of "dry leaves."

Subsequently, Patnala [17] developed a relatively simple and inexpensive extraction and iso‐ lation procedure for *Sceletium* alkaloids. In general, Sceletium plant powder was extracted using ethanol by soxhlet extraction followed by alcohol removal and acidification. Hexane was used to wash the acidic solution and the organic phase discarded. Subsequently, ammo‐ nia solution was used to neutralize and result in alkaline solution, and the latter was further extracted with dichloromethane (DCM). The DCM fractions were collected into a round‐ bottomed flask and evaporated under vacuum to yield a brown viscous liquid containing alkaloids. Following the separation of components by column chromatography, collected elu‐ ents were spotted on a TLC plate (**Figure 4**). The TLC plate was first observed under UV254 which showed extensive related substances (*acetone‐Track 3 and acetonitrile‐Track 4*) and fur‐ ther sprayed with Dragendorff's reagent (**Figure 4**). The acetone fraction and the acetonitrile (ACN) fractions were found to contain alkaloids.

The ACN fraction was tested for its UV spectrum which showed a maximum at 298.2 nm was found to be ∆<sup>7</sup> mesembrenone (**Figure 5**), and this fraction was further purified by preparative TLC.

In view of the fact that Sceletium species contain complex mixtures of closely related alkaloi‐ dal components, appropriate analytical methods for their separation and identification are

**Figure 4.** TLC plate of the column fractions by developed TLC method observed under UV254 and subsequently sprayed with Dragendorff's reagent for positive identification of alkaloids.

Sceletium Plant Species: Alkaloidal Components, Chemistry and Ethnopharmacology http://dx.doi.org/10.5772/66482 95

**Figure 5.** HPLC‐PDA of ACN fraction‐spectrum index plot (top) and chromatogram (bottom).

essential prerequisites for chemotaxonomic profiling of these species. Furthermore, the avail‐ ability of relevant alkaloid reference standards is also necessary including the use of an ana‐ lytical method with required specificity for fingerprinting. These foregoing considerations are essential to facilitate the proper identification of *Sceletium* species based on a chemotaxonomic approach [11].

#### **5. Development of analytical methodologies for identification and quality control (QC) of** *Sceletium* **plant material and associated products**

#### **5.1. High‐performance liquid chromatography (HPLC)**

reported the extraction of alkaloids from *Sceletium namaquense* which yielded mesembrine,

some unidentified alkaloids. Smith et al. [15] extracted mesembrenol (Table 2, No. 9) {incor‐ rectly designated as 4′‐*O*‐demethylmesembrenol and labeled (1) in their paper}, mesembrine and mesembrenone from Sceletium plant material. Gericke et al. [16] in their US patent appli‐ cation described the extraction of mesembrine‐type alkaloids with a yield of between 15 and

Subsequently, Patnala [17] developed a relatively simple and inexpensive extraction and iso‐ lation procedure for *Sceletium* alkaloids. In general, Sceletium plant powder was extracted using ethanol by soxhlet extraction followed by alcohol removal and acidification. Hexane was used to wash the acidic solution and the organic phase discarded. Subsequently, ammo‐ nia solution was used to neutralize and result in alkaline solution, and the latter was further extracted with dichloromethane (DCM). The DCM fractions were collected into a round‐ bottomed flask and evaporated under vacuum to yield a brown viscous liquid containing alkaloids. Following the separation of components by column chromatography, collected elu‐ ents were spotted on a TLC plate (**Figure 4**). The TLC plate was first observed under UV254 which showed extensive related substances (*acetone‐Track 3 and acetonitrile‐Track 4*) and fur‐ ther sprayed with Dragendorff's reagent (**Figure 4**). The acetone fraction and the acetonitrile

The ACN fraction was tested for its UV spectrum which showed a maximum at 298.2 nm was

In view of the fact that Sceletium species contain complex mixtures of closely related alkaloi‐ dal components, appropriate analytical methods for their separation and identification are

**Figure 4.** TLC plate of the column fractions by developed TLC method observed under UV254 and subsequently sprayed

mesembrenone (**Figure 5**), and this fraction was further purified by preparative

mesembrenone, tortuosamine and

mesembrenone, *Sceletium* A4, N‐formyltortuosamine, ∆<sup>7</sup>

94 Alkaloids – Alternatives in Synthesis, Modification and Application

(ACN) fractions were found to contain alkaloids.

with Dragendorff's reagent for positive identification of alkaloids.

35 mg per gram of "dry leaves."

found to be ∆<sup>7</sup>

TLC.

Chromatographic fingerprinting has been widely accepted and recommended by various regulatory authorities such as WHO [18], US‐FDA [19] and EMEA [20] to assess the consis‐ tency of batch to batch dosage forms containing phytochemical components of the harvested plants. In the current international regulatory scenario, qualitative and quantitative analytical methods are considered mandatory.

Validated analytical methods to assay *Sceletium* plant material and dosage forms for relevant alkaloidal content were reported for the first time where a simple, accurate, precise, rapid and

**Figure 6.** HPLC chromatogram of relevant standard Sceletium alkaloids.

reproducible HPLC method was developed for the identification and quantitative analysis of five relevant *Sceletium* alkaloids, Δ<sup>7</sup> mesembrenone, mesembranol, mesembrenone, mesem‐ brine and epimesembranol. This method has also been successfully used to study chemo‐ taxonomy of some *Sceletium* species and has provided impetus for the future development of quality monographs for plant and dosage forms containing *Sceletium* [21]. Subsequently, this method has been applied for the identification [22] and quantization of two additional alkaloids: Sceletium A4 and mesembrenol. **Figure 6** illustrates the chromatographic profile of the above‐mentioned alkaloids.

#### **5.2. LC‐MS/MS**

Since a number of variables including species differences, harvesting time, growing condi‐ tions, storage and processing contribute to the variation in phytochemical components in plants, it is therefore necessary to use appropriate and specific analytical methods to ensure quality which may affect the safety and efficacy of products prepared from plant material [23]. In particular, with respect to the *Sceletium* plant species which contain closely related mesem‐ brine‐type compounds of which some are epimers and have isobaric chemistries [17], specific methods are necessary. Since HPLC using UV detection cannot discriminate between such compounds, detection by MS enhances the accuracy and specificity of the analytical method, thereby reducing the risk of using an inappropriate *Sceletium* species for the indications on the product label [22]. In addition, this method proved valuable to monitor the fermentation process of *Sceletium* plant material [24]. Hence, the current qualitative LCMS method, and concurrent application of the previously reported quantitative assay method [22], provides valuable analytical procedures for the identification and QC of *Sceletium* plant material and its dosage forms. The application of the LC‐ESI‐MS tandem mass spectroscopy provides unique fragmentation patterns which facilitates the identity of specific alkaloid in complex matrices and thus provides valuable confirmatory data for chemotaxonomic studies [11].

#### **5.3. Capillary zone electrophoresis (CZE)**

Since alkaloids are relatively strong bases in general [25], they are good candidates for CE analysis. A CZE method was developed and validated and applied to fingerprint the presence of alkaloids in a marketed tablet product containing Sceletium plant material [26].

#### **6. Ethnopharmacology**

reproducible HPLC method was developed for the identification and quantitative analysis of

brine and epimesembranol. This method has also been successfully used to study chemo‐ taxonomy of some *Sceletium* species and has provided impetus for the future development of quality monographs for plant and dosage forms containing *Sceletium* [21]. Subsequently, this method has been applied for the identification [22] and quantization of two additional alkaloids: Sceletium A4 and mesembrenol. **Figure 6** illustrates the chromatographic profile of

Since a number of variables including species differences, harvesting time, growing condi‐ tions, storage and processing contribute to the variation in phytochemical components in plants, it is therefore necessary to use appropriate and specific analytical methods to ensure quality which may affect the safety and efficacy of products prepared from plant material [23]. In particular, with respect to the *Sceletium* plant species which contain closely related mesem‐ brine‐type compounds of which some are epimers and have isobaric chemistries [17], specific methods are necessary. Since HPLC using UV detection cannot discriminate between such compounds, detection by MS enhances the accuracy and specificity of the analytical method, thereby reducing the risk of using an inappropriate *Sceletium* species for the indications on the product label [22]. In addition, this method proved valuable to monitor the fermentation process of *Sceletium* plant material [24]. Hence, the current qualitative LCMS method, and concurrent application of the previously reported quantitative assay method [22], provides valuable analytical procedures for the identification and QC of *Sceletium* plant material and its dosage forms. The application of the LC‐ESI‐MS tandem mass spectroscopy provides unique

mesembrenone, mesembranol, mesembrenone, mesem‐

five relevant *Sceletium* alkaloids, Δ<sup>7</sup>

**Figure 6.** HPLC chromatogram of relevant standard Sceletium alkaloids.

96 Alkaloids – Alternatives in Synthesis, Modification and Application

the above‐mentioned alkaloids.

**5.2. LC‐MS/MS**

The use of specific herbal medicines varies depending on specific regions and ethnopharma‐ cological experiences, which makes this form of treatment inconsistent. Safety and efficacy are major concerns due to poor documentation and a dearth of scientific research on this sub‐ ject. The World Health Organization (WHO) notes that of 119 plant‐derived pharmaceutical medicines, about 74% are used in modern medicine in ways that correlate directly with their traditional use as herbal medicines [27].

The traditional preparation of *Sceletium* known as "*Kougoed"* or "*Channa"* is a fermented prep‐ aration used by the native Bushmen of Namaqualand. Traditionally, its main use for its psy‐ choactive properties involved a prior fermentation by the Khoisan tribe of southern Africa, who purported that the psychoactive effect of this plant is greatly enhanced [2, 3]. Based on this perception, *Sceletium* plants and their products are marketed with claimed improve‐ ments in mood and reduction of anxiety, when the fermented plant material is used either by chewing or smoking. In general, the fermentation process involves crushing the whole plant material or aerial parts which are then placed in sealed containers for several days and dried under natural sunlight. Patnala [17, 24] subsequently confirmed that the fermentation process transforms mesembrine to ∆<sup>7</sup> mesembrenone and requires an aqueous environment together with the presence of light to facilitate such a transformation.

#### **7. Biological activities and medicinal properties of** *Sceletium alkaloids*

The study of the phytochemical composition of Sceletium was provoked as a result of anec‐ dotal information describing the use of these plants by early inhabitants of Southern Africa [28]. Typical examples of medicinal use have been described in the Ethnopharmacology sec‐ tion above. It can be gleaned from current scientific literature that several scientific groups working on various aspects of Sceletium plants have focused on the biological activity of these alkaloids [29]. It should be noted that antidepressant activity of mesembrine‐type alkaloids has been demonstrated in animal models, of which, where mesembrine has been the principal alkaloid. The antidepressant activity is reportedly based on selective inhibition of serotonin reuptake, and mesembrine has a weak narcotic effect [30]. A recent study indicates that high‐ mesembrine Sceletium extract is a monoamine releasing agent, rather than only a selective serotonin reuptake inhibitor [31]. Zembrin® a marketed product containing a standardized extract of *Sceletium tortuosum* has been studied using human volunteers for its acute effects in the brain, and its pharmacological activity and potential therapeutic effect are reported to be based on the inhibiting reuptake of 5‐HT and PDE4. It is suggested that a 25 mg dose of Zembrin® has the potential of reducing anxiety in humans [32].

#### **8. Conclusions**

Although eight *Sceletium* plant species have been formally classified in accordance with usual botanic taxonomy, we have observed the existence of various subspecies related to the tor‐ tuosum‐type plants. Furthermore, the identified alkaloidal constituents vary between each of these plant species. In the tortuosum‐type plants, mesembrenone (**Table 2**, No. 8), where the double bond occurs between C4‐5; Δ7mesembrenone (**Table 3**, No. 15), where the double bond occurs between C7‐7a; and the epimers, mesembranol and epimesembranol, clearly have closely related chemical structures. Hence, accurate identification and characterization is nec‐ essary to confirm the true identity of each species [22] in view of the close similarity between such chemical structures. such relevant information provides invaluable data to confirm the true identity of each species. These "tortuosum"‐type Sceletium species contain mesembrine as the major alkaloid along with other minor alkaloids, ∆<sup>7</sup> mesembrenone, mesembrenone and mesembranol and clearly differ from the other species. However, a subspecies of tortuo‐ sum type, *S. strictum* contains mesembrenone as the major alkaloidal component alongside mesembrine [11]. The above‐mentioned information can be gleaned from published studies on *Sceletium* plants [17, 22, 24].

The advent and availability of modern instrumental techniques have provided valuable tools to identify differences between species based on phytochemical composition. Such approaches for taxonomical classification of plants and their species facilitate a superior and more accu‐ rate method which supersedes the classical techniques based on morphological aspects.

Although plants have been used for their medicinal properties for centuries relating back to biblical times, the interest and development of medicinal products containing plant material have grown exponentially where such products, often referred to a complementary medicines currently constitute and industry with sales of billions of dollars annually. However, there is growing concern relating to quality, safety and efficacy of such products where regulatory requirements relating to the provision of such necessary evidence currently leaves a lot to be desired and in instances have demonstrated undesirable risks to vulnerable users. Proper quality control requires the application of appropriate analytical techniques to assess the identity and quality of complementary medicines containing plant material. Quality control methods require access and availability to reference standards for each product which is mar‐ keted for medicinal use. As far as Sceletium‐based products are concerned, the information relating to isolation, identification, quantification and purification of individual alkaloidal compounds found in Sceletium species provides valuable data for use in the quality con‐ trol of medicines containing Sceletium plant material. While quality control is an essential component to ensure the quality of medicines, evidence of the safety and efficacy is further essential components, and it is important that such data are generated through clinical trials in humans. Furthermore, the absorption, distribution, metabolism and elimination (ADME) of administered products and associated kinetics should be studied. Such studies require the development and validation of appropriate analytical techniques to monitor the active ingredient(s) and the resulting metabolite(s) where applicable.

Modern instrumental methods such HPLC, LC‐MS, CZE and associated analytical technolo‐ gies have been invaluable in developing profiles for fingerprinting, identification and charac‐ terization of the relevant alkaloids and their specific plant associations as well as serving as an important tool for QC purposes of plant material and herbal medicines containing *Sceletium*. In addition, such techniques are also necessary to study the safety, efficacy, ADME and kinet‐ ics of medicinal products containing plant material.

#### **Author details**

extract of *Sceletium tortuosum* has been studied using human volunteers for its acute effects in the brain, and its pharmacological activity and potential therapeutic effect are reported to be based on the inhibiting reuptake of 5‐HT and PDE4. It is suggested that a 25 mg dose of

Although eight *Sceletium* plant species have been formally classified in accordance with usual botanic taxonomy, we have observed the existence of various subspecies related to the tor‐ tuosum‐type plants. Furthermore, the identified alkaloidal constituents vary between each of these plant species. In the tortuosum‐type plants, mesembrenone (**Table 2**, No. 8), where the double bond occurs between C4‐5; Δ7mesembrenone (**Table 3**, No. 15), where the double bond occurs between C7‐7a; and the epimers, mesembranol and epimesembranol, clearly have closely related chemical structures. Hence, accurate identification and characterization is nec‐ essary to confirm the true identity of each species [22] in view of the close similarity between such chemical structures. such relevant information provides invaluable data to confirm the true identity of each species. These "tortuosum"‐type Sceletium species contain mesembrine

and mesembranol and clearly differ from the other species. However, a subspecies of tortuo‐ sum type, *S. strictum* contains mesembrenone as the major alkaloidal component alongside mesembrine [11]. The above‐mentioned information can be gleaned from published studies

The advent and availability of modern instrumental techniques have provided valuable tools to identify differences between species based on phytochemical composition. Such approaches for taxonomical classification of plants and their species facilitate a superior and more accu‐ rate method which supersedes the classical techniques based on morphological aspects.

Although plants have been used for their medicinal properties for centuries relating back to biblical times, the interest and development of medicinal products containing plant material have grown exponentially where such products, often referred to a complementary medicines currently constitute and industry with sales of billions of dollars annually. However, there is growing concern relating to quality, safety and efficacy of such products where regulatory requirements relating to the provision of such necessary evidence currently leaves a lot to be desired and in instances have demonstrated undesirable risks to vulnerable users. Proper quality control requires the application of appropriate analytical techniques to assess the identity and quality of complementary medicines containing plant material. Quality control methods require access and availability to reference standards for each product which is mar‐ keted for medicinal use. As far as Sceletium‐based products are concerned, the information relating to isolation, identification, quantification and purification of individual alkaloidal compounds found in Sceletium species provides valuable data for use in the quality con‐ trol of medicines containing Sceletium plant material. While quality control is an essential component to ensure the quality of medicines, evidence of the safety and efficacy is further essential components, and it is important that such data are generated through clinical trials

mesembrenone, mesembrenone

Zembrin® has the potential of reducing anxiety in humans [32].

98 Alkaloids – Alternatives in Synthesis, Modification and Application

as the major alkaloid along with other minor alkaloids, ∆<sup>7</sup>

**8. Conclusions**

on *Sceletium* plants [17, 22, 24].

Srinivas Patnala<sup>1</sup> and Isadore Kanfer2, 3\*

\*Address all correspondence to: izzy.kanfer@gmail.com

1 Basic Sciences Research Centre, KLE University, Belgaum, India

2 Faculty of Pharmacy, Rhodes University, Grahamstown, South Africa

3 Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada

#### **References**


[21] Patnala S, Kanfer I. HPLC analysis of Mesembrine‐type alkaloids in Sceletium plant material used as an African traditional medicine. Journal of Pharmacy & Pharmaceutical Sciences 2010;**13(4)**:558‐570.

[8] Jeffs PW, Capps T, Johnson DB, Karle JM, Martin NH, Rauckman B. Sceletium alkaloids. VI. Minor alkaloids of S. namaquense and S. strictum. Journal of Organic Chemistry

[9] Arndt RR, Kruger PEJ. Alkaloids from Sceletium joubertii.bol. The structure of jouber‐

[10] Herbert RB, Kattah AE. The biosynthesis of Sceletium alkaloids in Sceletium subveluti‐

[11] Patnala S, Kanfer I. Chemotaxonomic studies of mesembrine‐type alkaloids in Sceletium

[12] Martin NH, Rosenthal D, Jeffs PW. Mass spectra of Sceltium alkaloids. Organic Mass

[13] Mesembrine. Merck Index. 13th edn. New Jersey: Merck Research Laboratories; 2001.

[14] Rimington C, Roets GCS. Notes upon the isolation of the alkaloidal constituent of the drug "Channa" or "Kougoed" (Mesebryanthemum anatomicum and M. tortuosum). Ondersteproot Journal of Veterinary Science and Animal Industry 1937;**9**:187‐191.

[15] Smith MT, Field CR, Crouch NR, Hirst M. The distribution of mesembrine alkaloids in selected TAXA of the mesembryanthemaceae and their modification in the sceletium

[16] Gericke NP, Van Wyk B‐E. Pharmaceutical Compositions Containing Mesembrine and Related Compounds. In US Patent: Gericke NP and Van Wyk B‐E. 09/194,836

[17] Patnala S. Pharmaceutical Analysis and Quality of Complementary Medicines: Sceletium

[18] General Guidelines for Methodologies on Research and Evaluation of Traditional Medicine. WHO/EDM/TRM/2000.1, Geneva: WHO; 2006; Annex1:21‐26. Available from: http://apps.who.int/iris/bitstream/10665/66783/1/WHO\_EDM\_TRM\_2000.1.pdf [Accessed:

[19] Guidelines for Dietary Supplements and Botanicals AOAC Official Methods of Analysis (2013) Appendix K, p. 2. Available from: http://www.eoma.aoac.org/app\_k.pdf

[20] Guideline on Specifications: Test Procedures and Acceptance Criteria for Herbal Substances, Herbal Preparations and Herbal Medicinal Products/Traditional Herbal Medicinal Products. 2006: EMEA/CVMP/815/00 Rev 1. European Medicines Agency (EMEA). Available from: http://www.ema.europa.eu/docs/en\_GB/document\_library/

Scientific\_guideline/2009/09/WC500003393.pdf [Accessed: 2016‐08‐29].

and Associated Products (Thesis). Grahamstown: Rhodes University; 2007.

tiamine and dehydrojoubertiamine. Tetrahedron Letters 1970;**37**:3237‐3240.

plant species. South African Journal of Science 2013;**109(3/4)**:882‐886.

derived 'Kougoed'. Pharmaceutical Biology 1998;**36(3)**:173‐179.

[US6,288,104B1]. 9‐11‐2006. South Africa; 1997.

num L. Bolus. Tetrahedron Letters 1990;**46(20)**:7105‐7118.

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100 Alkaloids – Alternatives in Synthesis, Modification and Application

Spectrometry 1976;**11**:1‐19.

2016‐11‐05].

[Accessed: 2016‐08‐29].


#### **Reducing of Alkaloid Contents During the Process of Lactic Acid Silaging Reducing of Alkaloid Contents During the Process of Lactic Acid Silaging**

Annett Gefrom and Annette Zeyner Annett Gefrom and Annette Zeyner

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66287

#### **Abstract**

The current study is conducted to investigate whether lupine grains can successfully be ensiled with residual moisture contents of around 65% and alkaloid contents can be reduced during the process of lactic acid formation by native and added lactic acid bacteria, respectively. Based on the fermentation quality of the sampled grain meal silage in the dry mass of 65% and further results, silaging is shown to be a suitable method of preservation. A reduction in the alkaloid content during the silaging cannot be assumed (statistical) due to the irregular dynamic of the observed content.

**Keywords:** lupines, lactic acid moist grain silaging, nutritional facts and alkaloid con‐ tent, lupine debittering

#### **1. Introduction**

At present, soy is the most important protein feed for monogastric farm animals and also of high importance for polygastric species, particularly in face of the ban of feedstuffs of animal origin. To cover the high demand for animal nutrition, the European Union is dependent on the import of soybeans and soy products. The European agricultural policy is focused on alternative protein sources because of the ongoing debate on the import of genetically modified soy. Additional arguments for maximum self‐supply with protein from local sources are the goals of sustainable production, the fluctuation of prices in soy bean business, and the future competition between food and energy production.

Legume grains like those from lupine species are valuable feedstuffs in this concern. Beside the remarkable protein content, they impress by a high density of energy.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Due to the uneven ripeness of the stock (**Figure 1**) and particularly if harvested at moist conditions, for example, in late summertime, a cost‐intensive artificial drying of the grains is necessary. Storing lupine grains with a residual moisture above 14% might increase the risk for elevated counts of moulds and yeasts.

**Figure 1.** Uneven ripeness of lupine corns (picture: A. Gefrom, LFA MV).

The chemical conservation with soda lye, propionic acid or feed urea is possible at a moisture of <20–25%, but demands a Hazard Analysis and Critical Control Concept. The material may be storable up to 6 months but might be limited in their use with respect to the target animal species or specific situation in question. Furthermore, chemical conservation of feedstuffs is not permitted for organic farming.

Ensiling lupine grains may be an inexpensive and ecologically advantageous alternative to produce a high‐protein feed of local origin that can be used in both conventional and organic farming. Lactic acid fermentation of fully mature legume grains with accordingly high dry matter contents is, however, impossible because the osmotolerance of the vast majority of lactobacilli is overused under such conditions. To create good conditions for silaging, lupine grains can be harvested either fully mature and remoistened or before full ripeness with accordingly high moisture content. According to technical aspects, harvesting before full ripeness and conservation by lactobacilli have the following particular benefits:


**•** Minimisation of nutrient loss (storage and threshing loss).

Due to the uneven ripeness of the stock (**Figure 1**) and particularly if harvested at moist conditions, for example, in late summertime, a cost‐intensive artificial drying of the grains is necessary. Storing lupine grains with a residual moisture above 14% might increase the risk

The chemical conservation with soda lye, propionic acid or feed urea is possible at a moisture of <20–25%, but demands a Hazard Analysis and Critical Control Concept. The material may be storable up to 6 months but might be limited in their use with respect to the target animal species or specific situation in question. Furthermore, chemical conservation of feedstuffs is

Ensiling lupine grains may be an inexpensive and ecologically advantageous alternative to produce a high‐protein feed of local origin that can be used in both conventional and organic farming. Lactic acid fermentation of fully mature legume grains with accordingly high dry matter contents is, however, impossible because the osmotolerance of the vast majority of lactobacilli is overused under such conditions. To create good conditions for silaging, lupine grains can be harvested either fully mature and remoistened or before full ripeness with accordingly high moisture content. According to technical aspects, harvesting before full

ripeness and conservation by lactobacilli have the following particular benefits:

**•** Early field clearance and therefore effective use of the land.

**•** Preservation of the feed can be performed by the farmer himself directly after harvest.

**•** Independence of harvest date and dry matter content (possible with moisture contents of

for elevated counts of moulds and yeasts.

104 Alkaloids – Alternatives in Synthesis, Modification and Application

**Figure 1.** Uneven ripeness of lupine corns (picture: A. Gefrom, LFA MV).

not permitted for organic farming.

up to about 35%).


The current study was conducted to investigate whether lupine grains can successfully be ensiled with residual moisture contents of around 65%, and alkaloid contents can be reduced during the process of lactic acid formation by native and added lactic acid bacteria, respectively. Hypotheses were that high-quality silages with reduced alkaloid contents can be produced in this way.

Alkaloids are important anti-nutritional factors limiting the use of lupine grains in the nutrition of farm animals. Due to the very effective alkaloids, low dose could induce physiological effects. Alkaloids damage among others the central nervous system, respiratory tract, and liver [1, 2]. Because of that, native bitter lupine grains are not suitable as feedstuff in particular for monogastric animals. For sweet lupine varieties provided as feed, actual guidelines demand a maximum alkaloid content of 0.05% to exclude intoxication [3, 4]. Further, the bitter taste can lead to a reduced acceptance and feed intake thus causing decreased performance. Pigs are particularly sensitive in this concern. However, under the environmental conditions of high temperatures, the accumulation of alkaloids may vary, and the alkaloid content in sweet lupines may be higher than the alkaloid limit for using lupines as feedstuff [5].

On the other hand, alkaloids are part of the protective system of the plant against pathogens. Alkaloids are nitrogenous combinations and attributive to some of the most toxic plant ingredients. Because of the molecular structure, the alkaloids in lupines allocate to chinolizidine alkaloide [6–8]. In corns of domestic lupines appear predominantly main quinolizidine alkaloids like spartein, lupinin, and lupanin in aggregate relevant to the lupine genre [9]. Other alkaloid contents in lupines are under 1%.


The alkaloids differ in toxicity. Spartein has a lower LD50 (median lethal dose) as lupanin [10]. Chinolizidine alkaloids are located are located in all plant organs, but the concentration and variation differ in texture, seasonal and daytime [9, 11, 12]. They are accumulated in corns and converted to proteins during maturation. They were also used as nitrogen source during germination [13–15].

It is possible that especially at not determinated varieties, green corns of the bastard branch have higher alkaloid content. Under the environmental conditions of high temperatures, the accumulation of alkaloids may vary, and the alkaloid content in sweet lupines may be higher than the alkaloid limit for using lupines as feedstuff [5, 16–18]. A post‐harvesting reduction in alkaloids through silaging may allow future cropping decisions basing more on phytosanitary conditions. The aim of this work was to examine whether alkaloids can be reduced in their concentration by fermentation with lactobacilli on the precondition that preservation process per se succeeds.

Alkaloids can be reduced by hydrolytic and oxidative enzymes during germination [13]. From a theoretical point of view, alkaloids may be decomposed throughout silaging under the action of native and supplemented lactic acid bacteria, respectively. A comprehensive action of plant and microbacterial enzymes is possible due to the grinding of the grains, the setting of suitable osmotic conditions, and a sufficiently long fermentation time.

The moisture found in grain meal allows enzymes to process the substances in suspension freed in the fluid phase of fermentation. Phytonutrients exist in the wet phase in soluble form and change through plant enzymes and enzymes of microorganisms occurring during fermentation [19, 20], so that phytonutrients can turn to low molecular weight units and into milk acid [21].

It has been demonstrated that the alkaloid content of lupine grains in milk ripeness can be reduced through silaging [22]. On the contrary, only small success in this concern was reported following silaging of the whole bitter lupine plant where the alkaloids seemed to be fairly stable in the acid milieu of the silage. It is assumed that alkaloids inhibited lactic acid bacteria during the silaging process [23].

Hopper et al. [24, 25] and Santana et al. [20], however, described a possible elimination of bitterness in lupines, since alkaloids serve as energy source for microorganisms. In this way, Toczko [26] and Toczko et al. [27] demonstrated the conversion of lupanin through Pseudo‐ monas sp. These bacteria are representatives for natural epiphytics which are proposed to be particularly important in the first phase of the process of fermentation. Santana and Empis [28] reduced the alkaloid content in flour of white bitter lupine seeds by 50% by incubation with Pseudomonas for 4 days. Camacho et al. [29] also demonstrated a reduction in alkaloids in lupine grains by incubation with *Lactobacillus acidophilus* (B‐1910) in a 12% total solids suspen‐ sion.

Apart from the possible reduction in alkaloids during fermentation, further benefits like physiologically effects of lactic acid silages of moist lupine grains are as follows:


### **2. Reducing of alkaloid contents during the process of lactic acid silaging**

#### **2.1. Material and method**

than the alkaloid limit for using lupines as feedstuff [5, 16–18]. A post‐harvesting reduction in alkaloids through silaging may allow future cropping decisions basing more on phytosanitary conditions. The aim of this work was to examine whether alkaloids can be reduced in their concentration by fermentation with lactobacilli on the precondition that preservation process

Alkaloids can be reduced by hydrolytic and oxidative enzymes during germination [13]. From a theoretical point of view, alkaloids may be decomposed throughout silaging under the action of native and supplemented lactic acid bacteria, respectively. A comprehensive action of plant and microbacterial enzymes is possible due to the grinding of the grains, the setting of suitable

The moisture found in grain meal allows enzymes to process the substances in suspension freed in the fluid phase of fermentation. Phytonutrients exist in the wet phase in soluble form and change through plant enzymes and enzymes of microorganisms occurring during fermentation [19, 20], so that phytonutrients can turn to low molecular weight units and into

It has been demonstrated that the alkaloid content of lupine grains in milk ripeness can be reduced through silaging [22]. On the contrary, only small success in this concern was reported following silaging of the whole bitter lupine plant where the alkaloids seemed to be fairly stable in the acid milieu of the silage. It is assumed that alkaloids inhibited lactic acid bacteria

Hopper et al. [24, 25] and Santana et al. [20], however, described a possible elimination of bitterness in lupines, since alkaloids serve as energy source for microorganisms. In this way, Toczko [26] and Toczko et al. [27] demonstrated the conversion of lupanin through Pseudo‐ monas sp. These bacteria are representatives for natural epiphytics which are proposed to be particularly important in the first phase of the process of fermentation. Santana and Empis [28] reduced the alkaloid content in flour of white bitter lupine seeds by 50% by incubation with Pseudomonas for 4 days. Camacho et al. [29] also demonstrated a reduction in alkaloids in lupine grains by incubation with *Lactobacillus acidophilus* (B‐1910) in a 12% total solids suspen‐

Apart from the possible reduction in alkaloids during fermentation, further benefits like

**•** The positive effects of lactic acid in terms of nutrition physiology, for example, the acidifi‐ cation of distinguished parts of the digestive tract to prevent the proliferation of clostridia

**•** The improvement in the feeding value due to the reduction in other anti‐nutritional factors

**•** Elevated contents of essential amino acids following proteolysis but only when desmolysis

physiologically effects of lactic acid silages of moist lupine grains are as follows:

osmotic conditions, and a sufficiently long fermentation time.

106 Alkaloids – Alternatives in Synthesis, Modification and Application

per se succeeds.

milk acid [21].

sion.

during the silaging process [23].

and other pathogenic microorganism.

**•** Improved intake and digestion of the feed [33].

such as oligosaccharides [30, 31].

can be prevented [32].

The experiments were performed with ripe, storage‐dry lupine grains (variety of sweet lupines 'Bora' and bitter lupine 'Azuro'; 2002) as well as with legume grains from different years (2005 and 2006) with a high residual moisture content (~65% DM).

In crushed seeds (sieve 3 mm; **Figure 2**) of ripe, storage‐dry lupine grains and at ~65% DM harvested *Lupinus angustifolius* L. (variety of sweet lupines 'Bora', 'Borlu', and bitter lupine 'Azuro') were determined the nutrient contents according to the Weende feed analysis [35]. Crude starch and water‐soluble carbohydrates (fructose, glucose, sucrose, and galactose) were analysed by HPLC (Shimadzu‐Germany GmbH) [36].

**Figure 2.** Crushed lupine seeds (picture: A. Gefrom, LFA MV).

The material was ground by ball mill (swing mill 'MM 200' Retsch GmbH & Co. KG, 42781 Germany, grain size 0.01 mm, 5 min with frequency of 30/s) for analysis of starch and alkaloids. The total alkaloid content (quinolizidine alkaloids) was determined by GC‐MS [37]:


The preparation of model silage (ROMOS—Rostock model silages [38]; **Figure 3a** and **b**) with 65% DM (moistened ripe, storage‐dry lupines meal and ~65% DM harvested lupine corns) followed using various biological silage additives (600 g material per plastic bag (three repetitions per variation)):

**Figure 3.** (a and b) Vacuum sealer and ROMOS—Rostock model silages (picture: A. Gefrom, LFA MV).

**•** Control (without additive)

Preparation To weigh ca. 0.5 g sample in centrifuge tubes, add 20 ml 0.5 N salt acid homogenize and store for 16 h at

Analysis Alkaloids were analysed by GC‐MS (Shimadzu‐Germany GmbH, GC‐MS QP 2010), type of column: BP‐1

The preparation of model silage (ROMOS—Rostock model silages [38]; **Figure 3a** and **b**) with 65% DM (moistened ripe, storage‐dry lupines meal and ~65% DM harvested lupine corns) followed using various biological silage additives (600 g material per plastic bag (three

and rest is absorbed with 1 ml methanol; sample could be stored by −20°C

**Figure 3.** (a and b) Vacuum sealer and ROMOS—Rostock model silages (picture: A. Gefrom, LFA MV).

300°C; externer standard: lupanin and spartein

108 Alkaloids – Alternatives in Synthesis, Modification and Application

repetitions per variation)):

room temperatures; centrifugalize by 4000 U/min for 30 min and by 20°C; pipette overlap (determine volume); add 6 N NaOH for adjust pH‐Wert of 12 in overlap (mix); alkaloids were determinated with solid phase extraction with extrelut column (Merck) and dichlormethan as eluent: add 18 ml to extrelut column (determine volume); wait 15 min; elute with 3 × 20 ml dichlormethan; solvent was evaporated

(length: 30 m; diameter: 0.25 mm; thickness: 0.25 μm); split proportion: 50; carrier gas: helium; passage: 94 ml/min; start temperature:120°C; end temperature: 260°C; ion source: 230°C; interface temperature:

**•** Lactic acid bacteria (LAB, homofermentative, *Lactobacillus plantarum*; 3 × 105 cfu/g FM; DSM 8862, 8866)

By using a vacuum sealer, the bags got evacuated and sealed and afterwards stored at 20°C for 90 days (**Figure 4**). The incubation was followed by the pH‐measurement (WTW MultiCal pH 526, precision: 0.01), the analysis of ammonia nitrogen and the analysis of organic acids and alcohol by HPLC and gas chromatograph (Shimadzu) in the silage extracts (50 g silage + 200 ml aqua dest.; 15 h at 5°C). The contents of lactic acid, fatty acid, and alcohol were analysed by HPLC. Gas chromatograph with standard was used for analysis of acetic acid, butyric acid, propionic acid, and alcohol (ethanol, propanol, butanol, butandiol). Parameter settings of HPLC and gas cromatograph are as follows:


**Figure 4.** Model silages after 90 days (picture: A. Gefrom, LFA MV).

Evaluation of parameters occurs according to DLG [39].

In freeze‐dried silage material nutritional parameters were analysed according to the Weende feed analysis and alkaloids were analysis by GC‐MS [37].

For statistical analysis, the computer software SPSS 14 for Windows (SPSS, Chicago, IL, USA) was used. Duncan test was applied to examine mean differences for significance. The level of significance was preset at *p* < 0.05.

#### **3. Results**

#### **3.1. Nutrient contents of lupine seeds and silages after incubation of 90 days**

The high energy and protein contents of moist harvested lupine grains from this study are in accordance with those reported by key feed tables (**Tables 1** and **2**). According to Jansen et al. [40], White et al. [41], and Wrigley [42], the crude starch in lupine corns analysed with enzymatical method contains approximately 2.4% in DM and is lower than contents after analysis with polarimetric method used for declarations in DLG. These authors supposed that crude starch contents analysed with polarimetric method could include sugar and non‐starch polysaccharides.


1 DLG 2014 [43] nutrient digestibility ruminants and UDP (undegrable protein): DLG [44]. 2 Polarimetric method.

3 Analysed by HPLC with enzymatic method; DM: dry matter; LAB: lactic acid bacteria [*Lactobacillus plantarum* 3 × 105 cfu/g fresh matter (FM), DSM 8862, 8866]; MEpig: metabolizable energy for pigs; calculated according to the estimation equation from GfE [45] and digestibility from DLG [44].

4 AMEN poultry: apparent metabolizable energy for poultry (N‐corrected): calculated according WPSA [46]; NEL MJ/kg DM and MEcow MJ/kg DM: energy calculated according to DLG [44]; nXP: available crude protein calculated according to DLG [44]; RNB: ruminal N‐bilance, RNB = (XP–nXP)/6.25; UDP: undegrable protein.

**Table 1.** Contents of proximate nutrients and energy in grains of blue lupine from literature (mature seeds, in 100% DM) compared to experimental data with lupine seeds harvested at ~65% DM (blue lupine).


1 Analysed by HPLC; with enzymatic method; control: without additive; DM: dry matter; LAB: lactic acid bacteria [*Lactobacillus plantarum* 3 × 105 cfu/g fresh matter (FM), DSM 8862, 8866]; MEpig: metabolizable energy for pigs; calculated according to the estimation equation from GfE [45] and digestibility from DLG [44].

2 AMEN poultry: apparent metabolizable energy for poultry (N‐corrected): calculated according WPSA [46]; NEL MJ/kg DM and MEcow MJ/kg DM: energy calculated according to DLG [44]; WSC: water soluble carbohydrates: fructose, glucose, sucrose, galactose

a,bSignificant (*p* < 0.05) differences of means between variants starting material (lupine 'Bora') and silages.

**Table 2.** Contents of proximate nutrients and energy in grains of blue lupine harvested at ~65% DM and therefrom produced silages after incubation of 90 days.

The content of nutritional feed value parameters is, apart from the fermentable carbohydrates (WSC), not affected by the silaging process, and the high feed and energy content of legumes are maintained in the grain silage.

#### **3.2. Quality of silages**

Evaluation of parameters occurs according to DLG [39].

110 Alkaloids – Alternatives in Synthesis, Modification and Application

feed analysis and alkaloids were analysis by GC‐MS [37].

significance was preset at *p* < 0.05.

**3. Results**

polysaccharides.

AMENpoultry4

Polarimetric method.

1

2

3

4

In freeze‐dried silage material nutritional parameters were analysed according to the Weende

For statistical analysis, the computer software SPSS 14 for Windows (SPSS, Chicago, IL, USA) was used. Duncan test was applied to examine mean differences for significance. The level of

The high energy and protein contents of moist harvested lupine grains from this study are in accordance with those reported by key feed tables (**Tables 1** and **2**). According to Jansen et al. [40], White et al. [41], and Wrigley [42], the crude starch in lupine corns analysed with enzymatical method contains approximately 2.4% in DM and is lower than contents after analysis with polarimetric method used for declarations in DLG. These authors supposed that crude starch contents analysed with polarimetric method could include sugar and non‐starch

**DLG (2014)1 Starting material 'Bora',**

**'Borlu', 'Azuro'; 2005/06,** *n* **= 6**

2.2 ±0.2

**3.1. Nutrient contents of lupine seeds and silages after incubation of 90 days**

Crude ash [% DM] 3.6 3.7 ±0.2 Crude protein [% DM] 33.5 35.8 ±0.9

Crude fat [% DM] 5.5 6.2 ±0.7 Crude fibre [% DM] 16.3 15.0 ±1.8

Crude sugar [% DM] 5.6 4.0 ±1.0 NEL [MJ/kg DM] 8.92 8.95 ±0.09 MEcow [MJ/kg DM] 14.19 14.27 ±0.13 MEpig [MJ/kg DM] 15.3 14.4 ±0.19

DLG 2014 [43] nutrient digestibility ruminants and UDP (undegrable protein): DLG [44].

according to DLG [44]; RNB: ruminal N‐bilance, RNB = (XP–nXP)/6.25; UDP: undegrable protein.

DM) compared to experimental data with lupine seeds harvested at ~65% DM (blue lupine).

6.0 <sup>3</sup>

[MJ/kg DM] 8.8 8.02 ±0.36

Analysed by HPLC with enzymatic method; DM: dry matter; LAB: lactic acid bacteria [*Lactobacillus plantarum* 3 × 105 cfu/g fresh matter (FM), DSM 8862, 8866]; MEpig: metabolizable energy for pigs; calculated according to the estimation

AMEN poultry: apparent metabolizable energy for poultry (N‐corrected): calculated according WPSA [46]; NEL MJ/kg DM and MEcow MJ/kg DM: energy calculated according to DLG [44]; nXP: available crude protein calculated

**Table 1.** Contents of proximate nutrients and energy in grains of blue lupine from literature (mature seeds, in 100%

UDP [% XP] 20 – nXP [%] 21.9 – RNB [g N/kg] +19 –

Crude starch [% DM] <sup>2</sup>

equation from GfE [45] and digestibility from DLG [44].

To ensure optimal osmotic conditions for silaging, lupine grains can either be harvested as mature grains and remoistered immediately before being ensiled, or harvested immature and ensiled as harvested. Based on the fermentation quality of the sampled grain meal silage in the dry mass range of ~65% DM and further results, silaging is shown to be a suitable method of preservation (**Table 3**). Harvest‐moist grains may be stored without problems, even without the addition of silage additives. The addition of high‐performance lactobacilli ensures the fermentation through an earlier and more comprehensive production of lactic acid. The production of acetic acid and alcohol was reduced.


AA: acetic acid; CON: control (without additive); control without additive; DM: dry matter; LA: lactic acid; LAB: addition of lactic acid bacteria [*Lactobacillus plantarum* 3 × 105 cfu/g fresh matter (FM), DSM 8862, 8866]; n. a.: not analysed; NH3‐N: ammonia nitrogen; SM: starting material; ΣAL: alcohol (ethanol, propanol, butanol, butandiol). a, b Significant (*p* < 0.05) differences of means between variants.

**Table 3.** Fermentation parameters of silages from lupine grains (harvested at ~65% DM) 90 days of ensiling and ensiled with or without lactic acid bacteria (LAB) as additive (own data, means of all samples from 2005 ('Bora', 'Borlu', 'Azuro'; *n* = 18).


Control without additive; DM: dry matter; LAB: addition of lactic acid bacteria [*Lactobacillus plantarum* 3 × 105 cfu/g fresh matter (FM), DSM 8862, 8866]; SM: starting material; alkaloids: external standard at measurement with GC‐MS: lupanin and spartein; a, b significant (*p* < 0.05) differences of means between variants.

**Table 4.** Alkaloids in dry corn from year 2002 ('Azuro') and in moist harvested grains (2005) from sweet lupines ('Borlu' and 'Bora') and bitter lupine ('Azuro') and their silages after 90 days incubation.

#### **3.3. Alkaloids**

**DM pH LA AA ΣAL NH3-N [%] [% DM] [% N]**

cfu/g fresh matter (FM), DSM 8862, 8866]; n. a.: not

*n* **[%] [% DM]**

Silage Control 6 63.5 ±0.72 2.246 ±0.34

Silage Control 6 66.6 ±0.2 2.765 ±0.50

Silage Control 12 66.1 ±0.7 0.079b ±0.01

Silage Control 6 66.1 ±0.9 0.082b ±0.01

Silage Control 6 66.2 ±0.6 0.075b ±0.01

'Borlu' SM 3 67.2 ±0.0 0.116a ±0.03

'Bora'' SM 3 67.1 ±0.1 0.103a ±0.02

LAB 18 64.4 ±0.85 2.615 ±0.95

LAB 6 66.1 ±0.7 2.743 ±0.22

LAB 12 66.1 ±0.2 0.074b ±0.02

LAB 6 66.2 ±0.2 0.078b ±0.01

LAB 6 65.9 ±0.2 0.069b ±0.02

cfu/g

CON 66.3 ±0.6 4.8c ±0.3 2.5b ±1.0 0.5b ±0.2 0.3a ±0.2 1.0a ±0.1 LAB 66.1 ±0.4 4.2d ±0.1 5.3a ±0.4 0.6a ±0.1 0.1b ±0.0 0.7b ±0.1 AA: acetic acid; CON: control (without additive); control without additive; DM: dry matter; LA: lactic acid; LAB:

analysed; NH3‐N: ammonia nitrogen; SM: starting material; ΣAL: alcohol (ethanol, propanol, butanol, butandiol). a, b

**Table 3.** Fermentation parameters of silages from lupine grains (harvested at ~65% DM) 90 days of ensiling and ensiled with or without lactic acid bacteria (LAB) as additive (own data, means of all samples from 2005 ('Bora', 'Borlu',

**Variety DM Alkaloids**

Bitter lupine Dry seed 'Azuro' SM 1 65.0 ±0.30 2.289 ±1.09

Sweet lupine 'Borlu' + ''Bora' SM 6 67.2 ±0.1 0.109a ±0.02

Control without additive; DM: dry matter; LAB: addition of lactic acid bacteria [*Lactobacillus plantarum* 3 × 105

**Table 4.** Alkaloids in dry corn from year 2002 ('Azuro') and in moist harvested grains (2005) from sweet lupines

lupanin and spartein; a, b significant (*p* < 0.05) differences of means between variants.

('Borlu' and 'Bora') and bitter lupine ('Azuro') and their silages after 90 days incubation.

fresh matter (FM), DSM 8862, 8866]; SM: starting material; alkaloids: external standard at measurement with GC‐MS:

Moist grains SM 3 66.1 ±0.1 2.991 ±1.42

SM 66.5 ±0.6 5.9a ±0.1 n. a.

112 Alkaloids – Alternatives in Synthesis, Modification and Application

addition of lactic acid bacteria [*Lactobacillus plantarum* 3 × 105

Significant (*p* < 0.05) differences of means between variants.

'Azuro'; *n* = 18).

**Table 4** shows the alkaloid content of lupine seeds (bitter 'Azuro' and sweet variants 'Bora') harvested at usual dry matter (2002) and approximate 65% DM (2005) and their silages after incubation of 90 days. The alkaloid content in analysed corns of bitter lupines ('Azuro') is higher than sweet lupines. Bitter lupines can contain high alkaloid contents of 4.5% DM [47, 48]. According to studies at the Julius Kühn Institute, the variety of the bitter lupine 'Azuro' contains 1.375% alkaloids in DM [29]. Sujak et al. [49] published a variety of alkaloids in sweet lupines between 0.05 and 0.24% in DM. Jansen et al. [16] published alkaloid contents in blue sweet lupines between 0006 and 0068% in DM. But under the environmental conditions of high temperatures, the accumulation of alkaloids may vary, and the alkaloid content in sweet lupines may be higher than the alkaloid limit for using lupines as feedstuff [5, 16, 17]. In blue sweet lupines 'Borlu' the alkaloid content reached a high level of 0.116% in DM and illustrated the variability between years. Otherwise, the grains were harvested earlier than normal, and the crop could contain more green corns with maybe high alkaloid content.

Against the statement of Camacho et al. [29], a reduction in alkaloid content in lupine silages through the fermentation process could not initially be shown because of the low differences and irregular dynamic in alkaloid contents. In all likelihood, the alkaloids are stabile in an acid environment [23]. A change in toxic potential of alkaloids in lupines after silaging should be reviewed in experimental feeding tests.

Pearson and Carr [50], Godfrey et al. [51], Bellof and Sieghart [52], and Allen [53] defined an experimental limit of tolerance of alkaloid contents in ration for feeding monogastric animals of 0.03%. But also contents of 200 mg alkaloid/kg could reduce feeding intake and growth of the animals [50, 51]. Petersen and Schulz [54] analysed an alkaloid content of 0.01% that effected the feeding intake.

#### **4. Conclusions**

Making silages of moist legume grains could be a lower‐cost and ecological conservation process for the production of protein‐rich feed in conventional and organic farming.

From the results of the experiments with fermented grain meal with high moisture content, it is possible to draw the following conclusions:


In own studies, the alkaloid contents could decrease tendencially by additional different lactobaccilus. A possible change in toxicity of the alkaloids during fermentation should be investigated by feeding studies.

**8.** The fermentation of early‐harvested legume grains with high moisture content can therefore be recommended as a suitable method of conservation. With respect to the economic and organisational benefits when compared to the discussed methods of chemical conservation, the lactic acid fermentation of grains under anaerobic conditions conforms to the requirements of organic agriculture and is therefore also interesting for this branch. Silaging in plastic tubes is recommended (**Figure 5**).

**Figure 5.** Silaging in plastic tubes is recommended (picture: A. Priepke, LFA MV).

#### **Acknowledgements**

The study was financed by Bundesministerium für Ernährung und Landwirtschaft. We are grateful to the H. Wilhelm Schaumann Stiftung for funding a scholarship.

#### **Author details**

**4.** The preservative effect of acid formation is primarily dependent on the moisture content. In respect of the process safety in industrial conditions, moisture content of 35% for lactic

**6.** The content of nutritional feed value parameters is, apart from the fermentable carbohy‐ drates, not affected by the silaging process, and the high feed and energy content of

**7.** A reduction in the alkaloid content during the silaging cannot be assumed (statistical) due to the irregular dynamic of the observed content. The number of samples should be considered for future investigations. Maybe other bacillus could reduce alkaloid contents. In own studies, the alkaloid contents could decrease tendencially by additional different lactobaccilus. A possible change in toxicity of the alkaloids during fermentation should

**8.** The fermentation of early‐harvested legume grains with high moisture content can therefore be recommended as a suitable method of conservation. With respect to the economic and organisational benefits when compared to the discussed methods of chemical conservation, the lactic acid fermentation of grains under anaerobic conditions conforms to the requirements of organic agriculture and is therefore also interesting for

**5.** Lactic acid bacteria stabilize the ensiling process and the silage quality.

this branch. Silaging in plastic tubes is recommended (**Figure 5**).

**Figure 5.** Silaging in plastic tubes is recommended (picture: A. Priepke, LFA MV).

The study was financed by Bundesministerium für Ernährung und Landwirtschaft. We are grateful to the H. Wilhelm Schaumann Stiftung for funding a scholarship.

**Acknowledgements**

acid silage inputs is to be recommended.

114 Alkaloids – Alternatives in Synthesis, Modification and Application

be investigated by feeding studies.

legumes are maintained in the grain meal silage.

Annett Gefrom1\* and Annette Zeyner2

\*Address all correspondence to: annett.gefrom@gmx.de

1 State Research Centre of Agriculture and Fishery Mecklenburg‐Western Pomerania, Germany

2 Group Animal Nutrition, Faculty of Natural Sciences III, Institute of Agricultural and Nu‐ tritional Sciences, Martin Luther University Halle‐Wittenberg, Halle (Saale), Germany

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### *Edited by Vasil Georgiev and Atanas Pavlov*

The book Alkaloids - Alternatives in Synthesis, Modification, and Application collects several chapters written by distinguished scientists and recognized experts in their respective fields of research. The purpose of this book is to focus the attention of a broad range of students, researchers, and specialists on some innovative and highly perspective areas in alkaloid research. The book covers several topics, guiding the readers from the development of nonconventional biotechnologies for alternative production of valuable alkaloids, through the application of modern chemical methods of asymmetric synthesis for production of synthetic and semisynthetic alkaloid derivatives, medicinal application of alkaloids as anesthetics and pain-relief drugs, analytical techniques for alkaloid profiling and their application in chemotaxonomy, quality control and standardization of raw plant material, to the importance of the control and reduction of alkaloid contents during production of animal feedstuffs.

Alkaloids - Alternatives in Synthesis, Modification and Application

Alkaloids

Alternatives in Synthesis,

Modification and Application

*Edited by Vasil Georgiev and Atanas Pavlov*

Photo by pilotL39 / iStock