Herbal extracts have shown much promise in the proliferation and differentiation of hMSCs in many different studies. The origin of these herbal extracts is mainly from Chinese traditional medicine, Indian Ayurveda medicine, and other South East Asian and Middle Eastern traditional medicine practices. Herbal extracts contain a plethora of phytochemicals such as polyphenols, flavonoids, and other plant-derived chemicals which synergistically aid in treating diseases in traditional medicine methods. Not only individual herbal extracts but also mixtures of different herbal extracts have shown promising results in traditional medicine. Also, the different parts of medicinal herbs, such as roots, leaves, stem, and fruits, are used in preparations for the treatment of different diseases. The herb Tithonia diversifolia is a worthy example, an extract of it being used for the treatment of diabetes, diarrhea, menstrual pain, malaria, hematomas, hepatitis, hepatomas, and wound healing [35]. The proven ability of many herbal extracts to treat a range of diseases has captured the attention of modern scientists and preliminary research is being carried out using stem cells and other cell types to find herbal extracts that are suitable stimulants based on their promising results in traditional medicine. Since herbs grow naturally, their local availability is high and the preliminary production costs will presumably be lower than for recombinant growth factors. As these extracts are composed of naturally occurring medicinal herbs, which may be regularly consumed by local communities, these may cause minimum side effects and have lower toxicity than the current stimulants. Therefore, herbal remedies may be safe and affordable alternatives to highly expensive recombinant and synthetic stimulants.

Effects of herbal extracts on the differentiation and proliferation of hMSCs

The studies described in the following sections elaborate on such herbal remedies and their possible mechanisms of action on hMSCs.

Osteogenic effects of herbal extracts

A traditional Chinese herbal formula (ZD-1) was found to have stimulatory effects on the proliferation and inhibitory effects on mineralization of hMSCs through down-regulation of several osteogenic markers such as osteocalcin, BMP-2, and osteopontin in the late stages [36].

The dried root of Drynariae fortune, Rhizoma Drynariae, contains flavonoid and triterpenoid compounds and its use results in increased bone cell viability, intracellular total proteins, alkaline phosphatase, and acid phosphatase [37]. Naringin, which is the main component of the Rhizoma Drynariae extract, enhanced the proliferation and osteogenic differentiation of BM-derived hMSCs [38]. The regulation of β-catenin, which is involved in osteoblastogenesis via Akt (protein kinase B) and AMPK (AMP activated protein kinase) signaling, has been demonstrated as a possible mechanism for the osteogenic properties of naringin [39].

An ethanol extract of the fruit of Ligustrum lucidum demonstrated an inhibitory activity on the proliferation of BM-derived hMSCs in a dose-dependent manner and a cytotoxic effect at a concentration of ≥200 μg/mL [40]. Conversely, the same study illustrated accelerated osteogenic activity at two specific concentrations, 50 μg/mL and 75 μg/ml. A suggested mechanism of action is based on a significant increase in expression of osteogenesis stimulating genes, β-catenin, BMP-2, cyclin D1, MT1-MMP (membrane type-1 matrix metalloproteinase), osteoprotegerin, and TBX3(T-Box 3) [40].

Dried root extract of the Korean herb Dipsacus asper, Dipsaci Radix, is used in the treatment of bone fracture. A dichloromethane fraction of Dipsaci Radix demonstrated not only that the whole extract possessed the ability to enhance osteoblastic differentiation from BM-derived hMSCs, but that the isolated single compound (hedraganin-3-O-(2-O-acetyl)-α-L-arabinopyranoside) worked similarly. Researchers suggested that the enhanced osteoblast differentiation was due to induced alkaline phophatase activity and expression of bone-specific proteins (bone sialoprotein and osteocalcin) by both the crude Dipsaci Radix extract and the isolated compound [33].

Foeniculum vulgare has been used in traditional medicine to increase milk secretion, promote menstruation, facilitate birth, and alleviate the symptoms of dysmenorrhea, which are conditions involving estrogenic activity. Based on estrogenic activity, a study on an ethanol extract of F. vulgare dried root showed a significant increase of BM-derived hMSC proliferation and differentiation into osteoblasts. The antiosteoporotic activity was thought to be stimulated through estrogenic activity of trans-anatole, an abundant component of the F. vulgare extract [41].

Herba epimedii extract showed increased rates of osteogenic activity on human BM-derived hMSCs via enhanced mRNA expression of BMP-2, BMP-4, Runx2 (Runt-related transcription factor 2), beta-catenin, and cyclinD1, all of which are BMP or Wnt signaling pathway-related regulators. This elucidated that the flavonoids of this extract promote osteogenesis through the BMP or Wnt-signaling pathway [42].

Ferula gummosa is used in Iranian traditional medicine and has applications in treating different types of diseases. The ethanol root extract of F. gummosa was proven to proliferate and differentiate human BM-derived hMSCs into osteocytes, demonstrating increased alkaline phosphatase activity [43].

Anti-adipogenic effects of herbal extracts

A Chinese herbal remedy, Quzhisu, had inhibitory effects on the adipogenic differentiation of BM-derived hMSCs by reducing the expression of PPARγ (peroxisome proliferator activated receptor γ) mRNA [44].

Tithonia diversifolia demonstrated antioxidant properties, which may help in treating obesity, a condition associated with decreased antioxidant levels resulting in high levels of adipogenesis [35]. This study recorded significant antiadipogenic activity due to reduced levels of reactive oxygen species by treating adipose derived hMSCs with a T. diversifolia aqueous extract [35]. Increased levels of pAMPK (phosporylated 5′-adenosine monophoshate-activated protein kinase), a key regulating enzyme involved in adipocyte differentiation and maturation [45], were detected [35].

Aloe-emodin is an anthraquinone present in aloe latex and has proven inhibitory effects on adipocyte differentiation when hMSCs were induced with 3-isobutyl-1-methylxanthine (IBMX) for adipogenesis. In comparison with control cultures, hMSCs treated with aloe-emodin had reduced expression levels of mRNA for resistin, adiponectin, aP(2), lipoprotein lipase, PPARγ, and tumor necrosis factor-α, which influence adipogenic pathways [46].

Neurogenic effects of herbal extracts

BM-derived hMSCs treated with a 1 % acetic acid extract of Mucuna gigantea had high proliferative characteristics and exhibited higher expression of mRNA for nestin (a neural precursor marker) and β-III tubulin (an immature neuron marker), suggesting the importance of M. gigantea in neural differentiation. As the M. gigantea extract contains L-DOPA, which is a precursor for neurotransmitter dopamine, using it in nerve-related stem cell therapy would enhance treatment capacity [47].

The extract of Angelica sinensis (Danggui) dried root, known as Radix Angelica sinesis (RAS), decreased β-amyloid peptide-induced neurotoxicity and tau phosphorylation in cultured cortical neurons [48], suggesting it has applications in neurodegenerative disorders. Treatment with RAS extract showed significantly higher percentages of neural like cell differentiation from AD-hMSCs compared with hMSCs treated with butylated hydroxyanisole; a commonly used neuronal inducer [49]. Ferulic acid, a main component of the RAS extract, also has proven ability to inhibit neurotoxic β-amyloid peptide aggregation in animal models [50], but the effects on patients with neurodegenerative disorders are yet to be determined.

Salvia miltiorrhiza extract demonstrated positive effects on differentiating Wharton jelly-derived hMSCs into neural like cells with significant morphological changes. Strongly positive markers were observed for nestin, β-tubulin, neurofilament, and glial fibrillary acidic protein. Neurite outgrowth-promoting protein expression (a neural cell marker) was markedly increased in hMScs after induction with the S. miltiorrhiza extract [51].

Endothelial/vascular genesis and angiogenesis effects of herbal extracts

Olive leaf extract resulted in hMSCs differentiating into endothelial cells and further into tubular structures, which are required in angiogenesis and vasculogenesis processes, while the genes for vascular endothelial growth factor, PCAM, platelet derived growth factor receptor, and vascular endothelial growth factor receptor (VEGFR)-1, involved in endothelial differentiation, are increasingly expressed in olive leaf extract-treated cells [52].

The rhizome extract of Curcumin longa L contains the phytochemical curcumin as the main component, which has antioxidant and anti-inflammatory properties. As stem cells require an antioxidant mechanism to survive and repair cells, curcumin with its antioxidant properties becomes a potential candidate to be applied in stem cell therapy research. An ethanol extraction of curcumin dried rhizome resulted in enhanced proliferation and differentiation of AD-hMSCs into endothelial progenitor cells through increased expression of the cell surface markers CD34, CD133, and VEGFR2 [53].

Proliferative and other effects of herbal extracts

Dhanwantram kashaya, used in Ayurvedic medicine to stimulate growth and nerve regeneration, resulted in increased proliferation and delayed senescence of Wharton jelly-derived hMSCs [54]. Yet another Chinese herbal remedy, Dan-Qi-TongMai-Pian, prepared mainly using Astragalus and Salvia, resulted in negative apoptosis of BM-derived hMSCs via stimulating c-IAP-1/2 expression and restricting caspase-3 activation [55].

The leaf extract of Carica papaya was found to have an effect on improving thrombocyte counts in both human and murine models, which suggest C. papaya leaf extract as an invaluable potential therapeutic agent for diseases such as dengue. The C. papaya extract up-regulated the in vitro synthesis of thrombopoiesis-related cytokines interleukin-6 and stem cell factor by hMSCs isolated from exfoliated deciduous teeth [56].

Viscum album, also known as Korean mistletoe lectin, was investigated for cytotoxic and proliferative activities on placenta-derived hMSCs. V. album extract showed cytotoxicity on HepG2 cancer cells at low concentrations of 1–5 pg/ml but induced significant proliferative properties in naive placenta-derived hMSCs via autophagy mechanisms [57].

Beneficial effects of herbal extracts in scaffolds

In vivo studies have shown an extract of Cissus quadrangularis, known as the Asthisandhani (bone setter) in Indian traditional medicine, to have bone fracture-healing properties [58]. Scaffolds treated with a C. quadrangularis extract showed significant differences with regard to hMSC proliferation, attachment, and enhanced osteoblast differentiation properties compared with scaffolds not treated with the extract [59].

The extract of Terminalia bellirica contains gallic acid, with anti-oxidant and cytoprotective properties, as a major component. In a study on hydrogel composition for use in stem cell therapy, a T. bellirica extract was found to result in significantly higher rates of hMSC proliferation and cell attachment [60].

Adverse effects of herbal extracts

The root extract of Angelicae dahuricae has been used as an anti-inflammatory, analgesic, antipyretic, and antioxidant remedy in Chinese and Korean herbal medicine. Nevertheless, this did not produce any significant rates of proliferation in hMSCs derived from gingiva [61].

The dried root of Cimicifuga heracleifolia or Cimicifuga foetida is known as Cimicifugae Rhizoma. An extract of Cimicifugae Rhizoma possesses anti-inflammatory, analgesic, and antipyretic properties and also been suggested as a potential treatment for dental diseases. hMSCs derived from healthy gingival tissue were treated with an aqueous Cimicifugae Rhizoma extract which, at high concentrations (such as 100 and 1000 μg/ml) affected stem cell morphology and reduced cell viability, suggesting potential adverse effects if administered orally [62].

A study on a root extract of Asiasarum, suggested for treating oral diseases, showed reduced cell viability of hMSCs derived from gingiva at higher concentrations (such as 100 and 1000 μg/ml), suggesting adverse effects on oral tissues at high doses of the extract [63].

Current drawbacks of use of herbal extracts

Herbal extracts may show promising results in in vitro studies but to apply these natural products in stem cell therapy will require more quality research to provide a better understanding into their mechanisms of action and effective pathways. As a crude extract a herbal could display beneficial properties, but its efficacy may be reduced during the manufacturing processes when produced at a commercial scale. Also, different solvents used to prepare crude extracts may cause adverse effects when used in therapy. Decreased absorption problems may occur when administered orally or intravenously; hence, local administration would be required when transplantation is performed, which can result in an invasive, painful procedure for the patient.

Although herbal products are endorsed as an excellent alternative to synthetic interventions, clinical applications are challenging due to the variability and complexity of bioactive constituents present in herbal formulations. Genetic, environmental, and cultural factors as well as the process of preparation affect the quality and the quantity of bioactive constituents, which may lead to undesirable side effects and low bioactivities. A well-defined and constant composition of the active constituents is a prerequisite for clinical application. Hence, “standardization”, which entails a set of scientific evaluations that assure quality, efficacy, safety, and reproducibility, was introduced to ensure the safety of herbal formulations in the global market [64]. Proper standardization will minimize quality issues with herbal preparations and ensure safe application of these in stem cell therapy.