**3. Herbal extracts: possible pets' health allies**

In this context, it is known that IFN‐γ represents the main cytokine involved in the immune response [21], as well as a crucial element in the onset of impaired tissue homeostasis condi‐

These observations clearly reinforce the potential toxicity associated with chronic consumption of poultry bones and derivatives by pets and pave the way for a new concept of food sensiti‐ zation due to contaminant presence as main enhancers of inflammatory processes, which typically characterize skin and gastrointestinal diseases. Although the Food and Drug Administration [31] and World Health Organization [32] have recently established maximum residue limits in foods, antibiotic residues in foods may still be present [33] thus explaining the persistence of dermatological manifestations in many pets. Moreover, international laws do not impose an antibiotic concentration evaluation in bones and fat, which are considered

A wide number of scientific reports suggest the possible toxicity and harmfulness of OTC toward human and pet health as a consequence of the consumption of meat derived from

All of these data may explain why chicken proteins, widely considered hypoallergenic and highly effective from a dietary point of view, play an important role in the etiology of several inflammatory pathologies. It is worth noting that the similarities between these phenomena and food allergies, atopy, and Flea allergy dermatitis have been observed. In spite of the limited evidence that canine food allergy is suggested to resemble a type I hypersensitivity reaction to allergens ingested by food, it cannot be excluded that non‐IgE‐mediated food allergies may also occur. Although literature reports have been evidenced that the prevalence of food allergies in *dogs* and *cats* is still unknown, the impressive number of cases is not justified merely on the basis of increased allergy spreading in civilized societies. Furthermore, it has been observed that 25% of the *cats* with both chronic GI and skin problems do not clinically express food allergies, while the remaining 75% had only gastrointestinal problems. On the other hand, there are no data regarding food allergies related to gastrointestinal problems in *dogs*. In addition, food allergies can be often confused with pyoderma, pruritic exudative dermatitis

Based on our recent studies, we investigated the sera of 24 *dogs* with food‐adverse reactions, that is, itching, diarrhea, otitis, dermatitis, conjunctivitis, overnight fasting, vomiting, flatu‐ lence, interdigital pyoderma, and anal sacs repletion for the presence of any haptens which might be responsible for such conditions by means of an enzyme‐linked immunosorbent assay (ELISA) (FS0059, IDLABS™ Inc. Biotechnology, PO Box 1145, Station CSC, London ON N6A

Results indicated the presence of OTC and doxycycline in all animal sera. Although only eight out of 24 *dogs* (33%) showed antibiotic concentrations above the ELISA detection limit (7.5 ng/ ml or ppb), all the remaining *dogs* presented serum levels of both antibiotics. OTC serum levels ranged from 2.61 to 56.04 ng/ml (6.30 ± 2.12; mean ± standard error of the mean), whereas doxycycline serum levels ranged from 1.28 to 22.84 ng/ml (5.20 ± 0.89; mean ± standard error of the mean). Our preliminary clinical investigation further confirmed the haptenic toxic‐

tions, typically related to autoimmunity or chronic inflammation [22–30].

328 Superfood and Functional Food - An Overview of Their Processing and Utilization

inedible, thus making pet food dangerous for pet's health [34].

5K2, Canada) according to the manufacturer's instructions [12].

intensive farming [15, 35–40].

or "hot spots."

What differentiates common pet food from a functional pet food is the presence of a protein source free of any contaminants, for example, antibiotics and hormones (as happens in intensive farming) as well as the addition of antioxidants, minerals, trace elements, herbal extracts, and medical plants in order to, respectively, stabilize, preserve, and improve the whole nutritional profile of the food.

Many scientific studies clearly demonstrate the efficacy of functional herbal extracts or medical plants for disease prevention or treatment, to improve overall health status or even to delay aging [41].

Based on these observations, we recently studied the anti‐inflammatory and antitoxic activity of a well‐standardized mixed pool of herbal extracts, as part of a commercially available pet food diet, following an OTC challenge [42]. More in detail, the extracts within the pool were *Ascophyllum nodosum* (66.3%)*, Cucumis melo* (1.5%)*, Carica papaya* (3.1%)*, Aloe vera* (3.1%)*, Haematococcus pluvialis* (1.1%), *Curcuma longa* (2.3%)*, Camellia sinensis* (1.5%)*, Punica granatum* (1.5%)*, Piper nigrum* (0.6%)*, Polygonum cuspidatum* (1.5%)*, Echinacea purpurea* (3.1%), *Grifola frondosa* (6.3%)*,* and *Glycine max* (4.6%)*.*

As previously explained, OTC is able to induce a significant IFN‐γ release from human T lymphocyte and non‐T cell [9] *in vitro*. Here, we demonstrated that this significant release was significantly reduced after a 24‐h individual co‐incubation of human T lymphocyte and non‐ T cells with all aforementioned extracts with the only exception of *A. vera* (**Figure 3**).

Further, we reported the same antitoxic and anti‐inflammatory activity of these extracts, with the only exception of *C. papaya* and with *P. nigrum*, on canine T lymphocytes challenged with OTC (**Figure 4**).

The antitoxic and anti‐inflammatory activity exerted by the extracts represents a further proof of the usefulness of the addition of selected and standardized herbal extracts within a pet food possibly free of any contaminant. In this way, it is possible to achieve a functional pet food able to support and enhance standard pharmacological treatments in the presence of infections or inflammatory diseases.

However, it is worth noting that enzyme deficiencies or different metabolic pathways make some plants, for example, onions, leeks, garlic, and chives, toxic for *dogs* and *cats* but not for humans [43]. For instance, one of the toxic effects of these plants is the oxidative hemolysis, which results from the inability of the antioxidant metabolic pathways to counteract an excess of oxidants in the erythrocytes. The toxicological mechanism is the following: (1) oxidation of the exposed β‐93 cysteine residues present in the hemoglobin and consequent sulfhemoglobin formation, (2a) precipitation, aggregation, and binding of sulfhemoglobin to the cell mem‐ brane and formation of the Heinz bodies, and (2b) membrane cross‐linking reactions occurring and eccentrocyte formation, (3) erythrocyte fragility increase and consequent extravascular hemolysis, (4) decreased blood oxygen transportation capacity, and (5) impaired delivery of oxygen to the tissues [44–46].

**Figure 3. The effects of botanicals on cytokine production by human PBMCs.** (a) shows the gating on viable lympho‐ cytes (R1 in dot plot graph) based on FSC and SSC parameters (see Section 2); (b) represents the gating on TH lympho‐ cytes (CD3+ CD8‐ as R2 in the dot plot graph) and on non‐T cells (CD3‐ CD8‐ cells as R3 in the dot plot graph); and (c) shows the IFN‐γ and IL‐4 production in human TH lymphocytes and non‐T cells incubated with ad hoc medium de‐ rived from botanicals or from mixture (see Section 2). Cytokine production was evaluated as percentage of IFN‐γ (*y*‐ axis) and IL‐4 (*x*‐axis)‐producing cells. The percentage of IFN‐γ (upper left quadrant inside the dot plots) and IL‐4 (lower right quadrant inside the dot plots)‐producing CD4 T (R2) and non‐T (R3) cells is reported. The different cell incubations with ad hoc medium derived from botanicals or from mixture (see Section 2) are indicated on top of each graph. (d) reports the statistic representation of 10 experiments on human CD4+ T lymphocytes evaluated as percent‐ age of IFN‐γ‐producing cells, \* *p* < 0.05. The different cell incubations with ad hoc medium derived from botanicals or from mixture (see Section 2) are indicated on top of each column. The abbreviation "ctr" in (c) and (d) indicates the basal cytokine production by PMBCs stimulated by PMA and ionomycin and in the presence of the ad hoc medium based on the same solubilizing vehicle but free from the botanicals (see Section 2); specifically, ctr 1 (*Ascophyllum n*., *Carica p.*, *Aloe v*., *Cucumis m*., *Glycine m*., and *Grifola f*.), ctr 2 (*Echinacea p., Piper n*.), ctr 3 (*Haematococcus p*.), and ctr 4 (the mixture of all the botanicals) (with the permission of Hindawi) [42].

and eccentrocyte formation, (3) erythrocyte fragility increase and consequent extravascular hemolysis, (4) decreased blood oxygen transportation capacity, and (5) impaired delivery of

**Figure 3. The effects of botanicals on cytokine production by human PBMCs.** (a) shows the gating on viable lympho‐ cytes (R1 in dot plot graph) based on FSC and SSC parameters (see Section 2); (b) represents the gating on TH lympho‐

shows the IFN‐γ and IL‐4 production in human TH lymphocytes and non‐T cells incubated with ad hoc medium de‐ rived from botanicals or from mixture (see Section 2). Cytokine production was evaluated as percentage of IFN‐γ (*y*‐ axis) and IL‐4 (*x*‐axis)‐producing cells. The percentage of IFN‐γ (upper left quadrant inside the dot plots) and IL‐4 (lower right quadrant inside the dot plots)‐producing CD4 T (R2) and non‐T (R3) cells is reported. The different cell incubations with ad hoc medium derived from botanicals or from mixture (see Section 2) are indicated on top of each

from mixture (see Section 2) are indicated on top of each column. The abbreviation "ctr" in (c) and (d) indicates the basal cytokine production by PMBCs stimulated by PMA and ionomycin and in the presence of the ad hoc medium based on the same solubilizing vehicle but free from the botanicals (see Section 2); specifically, ctr 1 (*Ascophyllum n*., *Carica p.*, *Aloe v*., *Cucumis m*., *Glycine m*., and *Grifola f*.), ctr 2 (*Echinacea p., Piper n*.), ctr 3 (*Haematococcus p*.), and ctr 4 (the

CD8‐

*p* < 0.05. The different cell incubations with ad hoc medium derived from botanicals or

cells as R3 in the dot plot graph); and (c)

T lymphocytes evaluated as percent‐

as R2 in the dot plot graph) and on non‐T cells (CD3‐

graph. (d) reports the statistic representation of 10 experiments on human CD4+

mixture of all the botanicals) (with the permission of Hindawi) [42].

oxygen to the tissues [44–46].

330 Superfood and Functional Food - An Overview of Their Processing and Utilization

cytes (CD3+

CD8‐

age of IFN‐γ‐producing cells, \*

**Figure 4. The effects of botanicals on IFN‐γ production by canine PBMCs.** (a) shows the gating on viable lympho‐ cytes (R1 in dot plot graph) based on FSC and SSC parameters (see Section 2). (b) represents the gating on CD4+ T lym‐ phocytes (CD3+ CD8‐ as R2 in the dot plot graph). (c) reports the results from one representative experiment showing the percentage (the number in upper quadrant) of IFN‐γ‐producing canine CD4+ T lymphocytes gated on R2 (*y*‐axis); *x*‐axis indicates the SSC parameter (see Section 2). The different co‐incubations of cells with ad hoc medium or mixture (see Section 2) are indicated on the top. (d) shows the statistic representation of the IFN‐γ production by canine CD4+ T lymphocytes evaluated as percentage of IFN‐γ‐producing cells in 10 representative experiments, \* *p* < 0.05. The abbrevi‐ ation "ctr" in (c) and (d) indicates the basal IFN‐γ production by PMBCs stimulated by PMA and ionomycin and in the presence of the ad hoc medium based on the same solubilizing vehicle but free from the botanicals (see Section 2): specifically, ctr 1 (*Ascophyllum n., Carica p., Aloe v., Cucumis v., Glycine m*., and *Grifola f*.), ctr 2 (*Echinacea p., Piper n*.), and ctr 3 (*Haematococcus p*.) (with the permission of Hindawi) [42].

This phenomenon can be obviously exacerbated in the presence of heritable high erythrocyte‐ reduced glutathione and potassium concentrations or glucose‐6‐phosphate dehydrogenase deficiency or zinc deficiency [47, 48].

Thus, dietary supplements, home‐made or commercially available pet food containing some plants or herbal extracts, might transform a functional food into a poisoning food.
