**2. Effects of herbal extracts on human stem cells**

#### **2.1. Stimulatory effects of herbal extracts on human stem cells**

Our literature search for the use of herbal preparations to stimulate stem cell proliferation and differentiation in clinical trials resulted in no publications or records, explaining that this area of research is at its infancy harping on the vital necessity of this line of research. However, many studies have been reported on the use of animal models with end results of *in vitro* studies, cross-linking the above-mentioned research areas, suggesting that the impending phases of research would hopefully culminate in clinical trials, leading to natural products being marketed as commercial stem cell-stimulating agents.

which explains rejuvenation and immunomodulation, has listed the use of approximately 200 herbs [28] which could be investigated for their regeneration capacities on stem cells. *Medhya Rasayana,* an intellectual/retention rejuvenation therapy method in Ayurveda that consists of four herbal plants, could be used individually or in combination [30]. Studies on stem cells treated with *Medhya Rasayana* extracts have shown the expression of nestin on stem cells, an early neural stem cell marker [29], confirming the ability of *Medhya* herbs to treat disorders

Application of Herbal Medicine as Proliferation and Differentiation Effectors of Human Stem Cells

http://dx.doi.org/10.5772/intechopen.72711

91

A growing concern of ameliorating radiation-induced normal tissue injury is arising as it affects the well-being of cancer patients. Stem cell therapy is used to replace these cells and tissues, and many examples are elaborated in the review of Benderitter *et al*. [31]. Authors have reviewed a number of studies related to ameliorating radiation-induced myelopathy by transplanting neural stem cells to the spinal code [32], potential applications of transplanting salivary gland stem cells in patients with radiation-induced xerostomia [31], potential benefits of transplanting stem cells and biomaterial in animal models with osteoradionecrosis [33] and transplanting autologous fat drafts including adipose-derived stem cells to treat radiationinduced late skin complications [34]. As herbal extracts had proven their differentiation aiding capabilities in *in vitro* studies, they could act as stimulants to produce increased numbers of stem cells required for patient transplantations. The following figure illustrates the different sources of human mesenchymal stem cells (hMSCs) and their differentiation capabilities with advantages and disadvantages of herbal stimulants and synthetic stimulants (**Figure 1**) [8].

**Figure 1.** A glimpse of hMSC sources and their differentiation capabilities stimulated with herbal extracts or synthetic

stimulants (Courtesy: Udalamaththa et al. [8]).

related to the neural system by increasing the differentiation ability of stem cells.

There are several reviews published summarizing the effects of different herbal extracts and their isolated bioactive compounds on human and other mammalian stem cells isolated from different sources. Our review published in 2016 elaborates on osteogenic, anti-adipogenic, neurogenic, endothelial/vascular genesis, angiogenesis and proliferative effects of herbal extracts on human mesenchymal stem cells mostly confirmed by RNA expression studies [8]. Dried root of Korean herb *Dipsacus asper* had been used in Korean traditional medicine for the treatment of bone fracture and the crude extract, and an isolated compound from the herb hedraganin-3-O-(2-O-acetyl)-α-L-arabinopyranoside demonstrated the osteogenic differentiation ability on bone marrow-derived hMSCs via the upregulation of bone-specific proteins and alkaline phosphatase activity [19]. Aloe emodin, present in Aloe latex, showed anti-adipogenic activity on hMSCs by reducing expression levels of mRNAs (resistin, adiponectin, aP(2), lipoprotein lipase, PPARγ and tumour necrosis factor-α) involved in adipogenic pathways [20]. Treatment of adipose-derived hMSCs with dried root extract of *Angelica sinensis*, an herb used in traditional Chinese medicine, resulted in significantly higher differentiation of neural-like cells than a commonly used neural inducer, butylated hydroxyanisole [21]. The neuroprotective ability of the same extract was proven by decreased induced neurotoxicity in cultured cortical neurons, increasing the extract's value as a potential candidate in treating neurodegenerative disorders [22]. A patent was obtained for endothelial differentiation of hMSCs treated with olive leaf extract with overexpression of gene vascular endothelial growth factor, PCAM, platelet-derived growth factor receptor and vascular endothelial growth factor receptor (VEGFR)-1 [23]. An updated list of herbals and mechanism of actions on MSCs, as well as a list of phytochemicals (resveratrol, genistein, naringin, icariin) isolated from plant extracts, were presented in a similar review published in 2017 [24]. As elaborated here, all four isolated compounds had proven their ability to differentiate MSCs into osteoblasts and osteocytes, possibly through the Wnt signalling pathway, upregulating gene expression of RUNX2 and Sirt-1 genes [25–27]. Combined therapy of adipose-derived hMSCs with icariin showed significantly improved survival rates of hMSCs as well as increased expression of endothelial markers and smooth muscle markers in rat models with diabetes mellitus-induced erectile dysfunction (DMED) inhibiting oxidative stress via the regulation of PI3K/Akt-STAT3 signal pathway [28]. A previous review published in 2014 demonstrated the well-established link between herbal preparations used in Ayurveda for a wide array of disorders with their proliferation and differentiation effects which were utilized in similar capacities on stem cell differentiation and proliferation, providing scientific proof of thousands of years old Ayurvedic predictions and practices [29]. *Rasayana*, the branch of Ayurveda which explains rejuvenation and immunomodulation, has listed the use of approximately 200 herbs [28] which could be investigated for their regeneration capacities on stem cells. *Medhya Rasayana,* an intellectual/retention rejuvenation therapy method in Ayurveda that consists of four herbal plants, could be used individually or in combination [30]. Studies on stem cells treated with *Medhya Rasayana* extracts have shown the expression of nestin on stem cells, an early neural stem cell marker [29], confirming the ability of *Medhya* herbs to treat disorders related to the neural system by increasing the differentiation ability of stem cells.

**2. Effects of herbal extracts on human stem cells**

90 Herbal Medicine

marketed as commercial stem cell-stimulating agents.

**2.1. Stimulatory effects of herbal extracts on human stem cells**

Our literature search for the use of herbal preparations to stimulate stem cell proliferation and differentiation in clinical trials resulted in no publications or records, explaining that this area of research is at its infancy harping on the vital necessity of this line of research. However, many studies have been reported on the use of animal models with end results of *in vitro* studies, cross-linking the above-mentioned research areas, suggesting that the impending phases of research would hopefully culminate in clinical trials, leading to natural products being

There are several reviews published summarizing the effects of different herbal extracts and their isolated bioactive compounds on human and other mammalian stem cells isolated from different sources. Our review published in 2016 elaborates on osteogenic, anti-adipogenic, neurogenic, endothelial/vascular genesis, angiogenesis and proliferative effects of herbal extracts on human mesenchymal stem cells mostly confirmed by RNA expression studies [8]. Dried root of Korean herb *Dipsacus asper* had been used in Korean traditional medicine for the treatment of bone fracture and the crude extract, and an isolated compound from the herb hedraganin-3-O-(2-O-acetyl)-α-L-arabinopyranoside demonstrated the osteogenic differentiation ability on bone marrow-derived hMSCs via the upregulation of bone-specific proteins and alkaline phosphatase activity [19]. Aloe emodin, present in Aloe latex, showed anti-adipogenic activity on hMSCs by reducing expression levels of mRNAs (resistin, adiponectin, aP(2), lipoprotein lipase, PPARγ and tumour necrosis factor-α) involved in adipogenic pathways [20]. Treatment of adipose-derived hMSCs with dried root extract of *Angelica sinensis*, an herb used in traditional Chinese medicine, resulted in significantly higher differentiation of neural-like cells than a commonly used neural inducer, butylated hydroxyanisole [21]. The neuroprotective ability of the same extract was proven by decreased induced neurotoxicity in cultured cortical neurons, increasing the extract's value as a potential candidate in treating neurodegenerative disorders [22]. A patent was obtained for endothelial differentiation of hMSCs treated with olive leaf extract with overexpression of gene vascular endothelial growth factor, PCAM, platelet-derived growth factor receptor and vascular endothelial growth factor receptor (VEGFR)-1 [23]. An updated list of herbals and mechanism of actions on MSCs, as well as a list of phytochemicals (resveratrol, genistein, naringin, icariin) isolated from plant extracts, were presented in a similar review published in 2017 [24]. As elaborated here, all four isolated compounds had proven their ability to differentiate MSCs into osteoblasts and osteocytes, possibly through the Wnt signalling pathway, upregulating gene expression of RUNX2 and Sirt-1 genes [25–27]. Combined therapy of adipose-derived hMSCs with icariin showed significantly improved survival rates of hMSCs as well as increased expression of endothelial markers and smooth muscle markers in rat models with diabetes mellitus-induced erectile dysfunction (DMED) inhibiting oxidative stress via the regulation of PI3K/Akt-STAT3 signal pathway [28]. A previous review published in 2014 demonstrated the well-established link between herbal preparations used in Ayurveda for a wide array of disorders with their proliferation and differentiation effects which were utilized in similar capacities on stem cell differentiation and proliferation, providing scientific proof of thousands of years old Ayurvedic predictions and practices [29]. *Rasayana*, the branch of Ayurveda A growing concern of ameliorating radiation-induced normal tissue injury is arising as it affects the well-being of cancer patients. Stem cell therapy is used to replace these cells and tissues, and many examples are elaborated in the review of Benderitter *et al*. [31]. Authors have reviewed a number of studies related to ameliorating radiation-induced myelopathy by transplanting neural stem cells to the spinal code [32], potential applications of transplanting salivary gland stem cells in patients with radiation-induced xerostomia [31], potential benefits of transplanting stem cells and biomaterial in animal models with osteoradionecrosis [33] and transplanting autologous fat drafts including adipose-derived stem cells to treat radiationinduced late skin complications [34]. As herbal extracts had proven their differentiation aiding capabilities in *in vitro* studies, they could act as stimulants to produce increased numbers of stem cells required for patient transplantations. The following figure illustrates the different sources of human mesenchymal stem cells (hMSCs) and their differentiation capabilities with advantages and disadvantages of herbal stimulants and synthetic stimulants (**Figure 1**) [8].

**Figure 1.** A glimpse of hMSC sources and their differentiation capabilities stimulated with herbal extracts or synthetic stimulants (Courtesy: Udalamaththa et al. [8]).

Although most of the reported research was on hMSCs, haematopoietic stem cells (HSCs) are also being investigated for their properties of proliferation and differentiation when treated with herbal extracts and their isolated compounds. Proliferation, differentiation and *in vitro* expansion of healthy hHSCs are important as many haematological malignancies disrupt the healthy hHSC populations. A review that summarizes a wide range of research publications on the use of Chinese herbal medicine (CHM) to promote recovery after HSC transplantation had elaborated the positive results of herbal extracts from plants such as Sheng Di Huang (*Rehmannia glutinosa*), Bai Zhu (*Atractylodes macrocephala*), Ren Shen (*Panax*), Dang Shen (*Codonopsis pilosula*), Mai Men Dong (*Ophiopogon japonicus*), Dang Gui (*Angelica sinensis*), Tai Zi Shen (*Pseudostellaria heterophylla*), Huang Qi (*Astragalus membranaceus*) and Ejiao (*Equus asinus*) [35]. A study on autologous and allogenic HSC transplanted in patients with chronic granulocytic leukaemia, acute non-lymphocytic leukaemia and lymphoma were treated with CHM concluded that treating with CHM reduces complications of transplantations and promotes recovery of haematopoietic functions [36]. More research on various other HSC transplantations against haematological malignancies such as severe aplastic anaemia patients [37], patients with myelodysplastic syndrome [38] and acute paediatric leukaemia [39] were cited herein [35], which had given positive results on patient survival rates, reduction of complications and increasing functional properties of haematopoietic cells. However, most of these studies were based on a low number of samples; hence, the need to perform such studies in large populations arises in order to validate and standardize the CHM procedures. *In vitro* studies and animal model studies had also been reported on HSC proliferation and differentiation to gather more scientific evidence to support small local clinical trials performed in isolation in individual countries. EMSA eritin, a polyherbal formulation had increased proliferation of HSC in irradiated BALB/c mice *in vivo* and triggered differentiation into the lymphopoiesis lineages [40]. Inducing of proliferation and attenuating of apoptosis were observed when an immune-mediated aplastic anaemia mouse model was treated with a modified Chinese herbal formula prepared with Radix astragali, Radix *Angelicae sinensis* and *Coptis chinensis Franch* [41].

ditioned with high doses via significantly decreasing caspase-3 activity [44]. *Tinospora cordifolia* and *Withania somnifera*, two widely used herbs used in Ayurveda for rejuvenating and anti-ageing treatment, had shown increase in proliferation and inhibition of senescence in WJ-MSCs *in vitro* [45], suggesting that pretreatment with these herbals would aid in *in vivo* 

Application of Herbal Medicine as Proliferation and Differentiation Effectors of Human Stem Cells

http://dx.doi.org/10.5772/intechopen.72711

93

Cancer stem cells (CSCs), the cells which are capable of self-renewal and produce the heterogeneous lineage of cancer cells [46], has become the most complicated issue in cancer therapy. A number of studies were reported which resulted in the reduction of cancer cells with the treatment of isolated phytochemicals such as epigallocatechin-3-gallate (EGCG), curcumin, resveratrol, lycopene, pomegranate extracts, luteolin, genistein, piperin, β-carotene and sulforaphane [45]. Specifically, sulforaphane, a phytochemical isolated from broccoli, had apoptosis-inducing effects on pancreatic CSCs [47] and could target breast CSCs effec-

However, in this scenario, scientists are changing their approach in the search for natural products by trying to select herbal extracts and preparations known to be effective against cancers in traditional medicine. This approach would be advantageous for both ends of traditional medicine and modern therapeutics, as traditional medicine will have a chance of proving the remedies in a scientific platform and also the modern therapeutics would have the benefit of using time tested anticancer remedies rather than screening thousands of plant extracts for this purpose without any clues. A review on targeting CSCs using TCM remedies and their active compounds had elaborated several approaches of herbal remedies acting on CSCs. Reversion of drug resistance of CSCs, inducing cell death and inhibiting cell proliferation, inhibiting metastasis and targeting CSCs-related miRNAs are the explained methods of TCM remedies targeting CSCs [49]. Berberine liposomes, isolated from rhizome of *Coptis chinensis*, showed anticancer effects on human breast CSCs transplanted in nude mice by penetrating the cell membrane, accumulating in mitochondria of CSCs and resulting in reversion of drug resistance and apoptotic pathway inducing cell death and inhibiting cell proliferation [50]. Curcumin and epigallocatechin gallate (EGCG) had synergistically targeted breast CSCs by downregulating stemness genes and inducing differentiation of these into non-stem cells [51]. Prostate cancer metastasis had been reduced by a combination of quercetin, extracted from *Dysosma veitchii* and EGCG by reducing activity of LEF-1/TCF responsive receptor [52]. Honokiol, a lignan isolated from *Magnolia officinalis*, had inhibited renal cancer metastasis by regulating miR-141/ZEB2 signalling [53]. Triphala, a widely used formulation in Ayurveda, had shown anticancer properties on human colon cancer stem cells by p53-independent proliferation inhibition and apoptosis inducing [54]. Also, a Sri Lankan group of scientists had investigated on anticancer properties of gedunin, a major compound found in *Azadirachta indica*, which confirmed its apoptotic-inducing properties against human embryonal carci-

transplantation procedures.

noma cells—a cancer stem cell model [55].

tively [48].

**2.2. Inhibitory effects of herbal extracts on cancer stem cells**

Although stem cell therapy had boosted disease therapy into the next level of modern therapeutic medicine, a major limitation is their poor survival after transplantation into the host, which could be resolved by supplementing the microenvironment with vitamins and other antioxidants [29] and other preconditioning strategies such as exposure to hypoxic conditions, oxidative stress and heat shock treatments [42]. Scientists are studying natural plant extracts and their isolated compounds as alternatives to synthetic growth factors and other stimulants to precondition the microenvironment for the survival of stem cells *in vivo*, as there are many reports on the presence of a wide array of beneficial phytochemicals in plants. Pretreatment of adipose-derived hMSCs with *C. setidens* herbal extract had resulted in increased survival of hMSCs by inhibiting ROS-induced apoptosis, suggesting the suitability of the extract to prevent ROS-induced oxidative stress by regulating the oxidative stress-associated signalling pathway and suppressing the apoptosis-associated signal pathway [43]. Extract of *Origanum vulgare* had protected murine mesenchymal stem cells from oxidative stress when preconditioned with high doses via significantly decreasing caspase-3 activity [44]. *Tinospora cordifolia* and *Withania somnifera*, two widely used herbs used in Ayurveda for rejuvenating and anti-ageing treatment, had shown increase in proliferation and inhibition of senescence in WJ-MSCs *in vitro* [45], suggesting that pretreatment with these herbals would aid in *in vivo*  transplantation procedures.

#### **2.2. Inhibitory effects of herbal extracts on cancer stem cells**

Although most of the reported research was on hMSCs, haematopoietic stem cells (HSCs) are also being investigated for their properties of proliferation and differentiation when treated with herbal extracts and their isolated compounds. Proliferation, differentiation and *in vitro* expansion of healthy hHSCs are important as many haematological malignancies disrupt the healthy hHSC populations. A review that summarizes a wide range of research publications on the use of Chinese herbal medicine (CHM) to promote recovery after HSC transplantation had elaborated the positive results of herbal extracts from plants such as Sheng Di Huang (*Rehmannia glutinosa*), Bai Zhu (*Atractylodes macrocephala*), Ren Shen (*Panax*), Dang Shen (*Codonopsis pilosula*), Mai Men Dong (*Ophiopogon japonicus*), Dang Gui (*Angelica sinensis*), Tai Zi Shen (*Pseudostellaria heterophylla*), Huang Qi (*Astragalus membranaceus*) and Ejiao (*Equus asinus*) [35]. A study on autologous and allogenic HSC transplanted in patients with chronic granulocytic leukaemia, acute non-lymphocytic leukaemia and lymphoma were treated with CHM concluded that treating with CHM reduces complications of transplantations and promotes recovery of haematopoietic functions [36]. More research on various other HSC transplantations against haematological malignancies such as severe aplastic anaemia patients [37], patients with myelodysplastic syndrome [38] and acute paediatric leukaemia [39] were cited herein [35], which had given positive results on patient survival rates, reduction of complications and increasing functional properties of haematopoietic cells. However, most of these studies were based on a low number of samples; hence, the need to perform such studies in large populations arises in order to validate and standardize the CHM procedures. *In vitro* studies and animal model studies had also been reported on HSC proliferation and differentiation to gather more scientific evidence to support small local clinical trials performed in isolation in individual countries. EMSA eritin, a polyherbal formulation had increased proliferation of HSC in irradiated BALB/c mice *in vivo* and triggered differentiation into the lymphopoiesis lineages [40]. Inducing of proliferation and attenuating of apoptosis were observed when an immune-mediated aplastic anaemia mouse model was treated with a modified Chinese herbal formula prepared with Radix astragali, Radix *Angelicae sinensis* and *Coptis* 

Although stem cell therapy had boosted disease therapy into the next level of modern therapeutic medicine, a major limitation is their poor survival after transplantation into the host, which could be resolved by supplementing the microenvironment with vitamins and other antioxidants [29] and other preconditioning strategies such as exposure to hypoxic conditions, oxidative stress and heat shock treatments [42]. Scientists are studying natural plant extracts and their isolated compounds as alternatives to synthetic growth factors and other stimulants to precondition the microenvironment for the survival of stem cells *in vivo*, as there are many reports on the presence of a wide array of beneficial phytochemicals in plants. Pretreatment of adipose-derived hMSCs with *C. setidens* herbal extract had resulted in increased survival of hMSCs by inhibiting ROS-induced apoptosis, suggesting the suitability of the extract to prevent ROS-induced oxidative stress by regulating the oxidative stress-associated signalling pathway and suppressing the apoptosis-associated signal pathway [43]. Extract of *Origanum vulgare* had protected murine mesenchymal stem cells from oxidative stress when precon-

*chinensis Franch* [41].

92 Herbal Medicine

Cancer stem cells (CSCs), the cells which are capable of self-renewal and produce the heterogeneous lineage of cancer cells [46], has become the most complicated issue in cancer therapy. A number of studies were reported which resulted in the reduction of cancer cells with the treatment of isolated phytochemicals such as epigallocatechin-3-gallate (EGCG), curcumin, resveratrol, lycopene, pomegranate extracts, luteolin, genistein, piperin, β-carotene and sulforaphane [45]. Specifically, sulforaphane, a phytochemical isolated from broccoli, had apoptosis-inducing effects on pancreatic CSCs [47] and could target breast CSCs effectively [48].

However, in this scenario, scientists are changing their approach in the search for natural products by trying to select herbal extracts and preparations known to be effective against cancers in traditional medicine. This approach would be advantageous for both ends of traditional medicine and modern therapeutics, as traditional medicine will have a chance of proving the remedies in a scientific platform and also the modern therapeutics would have the benefit of using time tested anticancer remedies rather than screening thousands of plant extracts for this purpose without any clues. A review on targeting CSCs using TCM remedies and their active compounds had elaborated several approaches of herbal remedies acting on CSCs. Reversion of drug resistance of CSCs, inducing cell death and inhibiting cell proliferation, inhibiting metastasis and targeting CSCs-related miRNAs are the explained methods of TCM remedies targeting CSCs [49]. Berberine liposomes, isolated from rhizome of *Coptis chinensis*, showed anticancer effects on human breast CSCs transplanted in nude mice by penetrating the cell membrane, accumulating in mitochondria of CSCs and resulting in reversion of drug resistance and apoptotic pathway inducing cell death and inhibiting cell proliferation [50]. Curcumin and epigallocatechin gallate (EGCG) had synergistically targeted breast CSCs by downregulating stemness genes and inducing differentiation of these into non-stem cells [51]. Prostate cancer metastasis had been reduced by a combination of quercetin, extracted from *Dysosma veitchii* and EGCG by reducing activity of LEF-1/TCF responsive receptor [52]. Honokiol, a lignan isolated from *Magnolia officinalis*, had inhibited renal cancer metastasis by regulating miR-141/ZEB2 signalling [53]. Triphala, a widely used formulation in Ayurveda, had shown anticancer properties on human colon cancer stem cells by p53-independent proliferation inhibition and apoptosis inducing [54]. Also, a Sri Lankan group of scientists had investigated on anticancer properties of gedunin, a major compound found in *Azadirachta indica*, which confirmed its apoptotic-inducing properties against human embryonal carcinoma cells—a cancer stem cell model [55].
