Introduction

Withania somnifera (Linn.) Dun. is commonly known as “Ashwagandha”, Winter Cherry, Indian ginseng. It belongs to the family Solanaceae. 23 species of genus Withania are reported among which, W. somnifera (L.) has high medicinal value. It is one of the most valued medicinal plants in Ayurveda and other traditional systems of medicine and has been used for over 3000 years. W. somnifera is regarded as one of the most useful herbs having ‘Vata’ pacifying properties (Singh and Kumar 1998). It is widely used in traditional Indian medicine system for curing variety of diseases. It possesses adaptogenic, tonic analgesic, antipyretic, anti-inflammatory and abortifacient properties and is one of the most extensively used plants in various systems of medicine (Chopra et al. 1958). Clinical trials and animal research support the use of W. somnifera for hepatotoxicity (Bhattacharya et al. 2000a), anxiety (Bhattacharya et al. 2000b), cognitive (Bhattacharya et al. 1995), neurological disorders (Kuboyama et al. 2005), inflammation (Al-Hindawi et al. 1992), hyperlipidemia (Visavadiya and Narasimhacharya 2007) and Parkinson’s disease (Ahmad et al. 2005). The fruits of this plant are rich in saponins and can be used as a substitute for soaps. The leaves are also known to act as an insect repellent (Schmelze et al. 2008).

Both leaves and roots of the plant are used as drug. Steroidal lactones known as withanolides (a group of biologically active oxygenated ergostane type steroidal lactones) occur in both parts of the plant (Kaushik et al. Communicated). The production of withanolides in the plant could be monitored through seasonal changes or growth periods.

Several studies concerning the chemistry, biological properties and genetics of withanolides have been carried out. These compounds have been intensely investigated because of their pronounced anti-tumor properties and novel steroidal structure. Alkaloids constitute another major group of components which have been isolated from W. somnifera. A number of alkaloids have also been isolated from the roots of W. somnifera, among them withanine is the main alkaloid comprising of 38% of the total alkaloid material (Atal et al. 1975). The chief withanolides of W. somnifera species found in India are withaferin A and withanolide D. Both withaferin A and withanolide D show antitumor and cytotoxic activities (Yoshida et al. 1979).

Present review summarizes the phytochemical diversity and pharmacological advances reported in literature.

Description of the plant

A small or middle-sized under shrub, erect, grayish, branched, 30–150 cm high, with greenish or lurid yellow flowers. Fruits are a berry enclosed in the green persistent calyx, green when unripe, turns to orange red when mature. The fruit contains numerous small capsicums like seeds. Flowering occurs nearly throughout the year.

The shoots specially stem, veins and the calyx are covered with minute star-shaped hairs. Leaves are simple, ovate, petiolate, entire, exastipulate, acute, glabrous and up to 10 cm long and petioles are around 1.25 cm long. On vegetative shoots, the leaves are alternatively arranged and large while on floral branches, they are oppositely arranged in pairs of one large and one small leaf and arranged somewhat parallel, having a cymose cluster of 5–25 inconspicuous pale green flowers in their axil.

The roots are fairly long and tuberous (~20–30 cms and 6–12 mm in diameter) with a short stem and with few (2–3) lateral roots of slightly smaller size, straight, unbranched. They have buff to grayish-yellow outer surface with longitudinal wrinkles and soft, solid mass with scattered pores in the center. The roots taste bitter and acrid (John 2014).

Geographical distribution of the plant

Withania somnifera (Linn.) Dunal has a fairly wide geographic distribution. Besides the Indian subcontinent, it is widely distributed in dry subtropical regions along the shores of the Mediterranean Sea, South Africa, Israel, Italy, Pakistan, Afghanistan, Palestine, Egypt, Jordan, Morocco, Spain, Canary Island, Eastern Africa Congo, Madagascar and South Africa, representing extensive variations of soil, rainfall, temperature and altitude (Atal et al. 1975).

In India, it is cosmopolitan and grows throughout the drier parts and sub-tropical regions (Hooker 1885). The plant is widely distributed in Northwestern, Bombay, Gujarat, Rajasthan, Madhya Pradesh, and Uttar Pradesh, Punjab plains, which extends up to the mountain region of Punjab, Himachal Pradesh and Jammu.

Various morphotypes

Extensive study by Atal in India revealed various morphotypes. He reported, extreme degree of variability in W. somnifera regarding the morphological characteristics and growth habits of plants found in different parts of India and in plants from other countries (Atal et al. 1975).

Five different morphotypes have been identified in India by Atal, in 1975, the details of which are described as follows:

  • Morphotype I Plant are usually not more than 30 cm high; stems growing from crown vary from one to many in number. It grows as an annual crop. Cultivated exclusively in Madhya Pradesh (Center India) and yields roots of commercial value.

  • Morphotype II Plants are 0.6–1.5 m long, stem is single erect, grows from crown giving off branches above the ground level. This form grows in the sandy desert soil of Marwar, Pilani and some other parts of Rajasthan.

  • Morphotype III Plants are 0.6–1 m high, branching starts from 15 to 30 cm above ground level. It India, it grows in Chandigarh and some other mountainous areas of Punjab and Uttar Pradesh.

  • Morphotype IV Plants are 0.6–0.75 m tall, profusely branches near the ground. The plant was found growing near Delhi.

  • Morphotype V Plants are exceptionally tall; 1.2–2.1 m. Wild growth of this form was seen near Delhi and Ahmedabad. The plant prefers shady habitats and is found in or along hedges, sometimes extending into open.

Phytochemical investigations

Many compounds have been isolated so far from different chemotypes of W. somnifera. These include alcoholic, alkaloids and withanolides compounds.

Alcoholic compounds

The earliest report available on the phytochemistry of the plant is by Power and Salway (1911) that studied the chemical principles of W. somnifera and reported the presence of a number of compounds from the roots and leaves of the plant. They reported two new monohydric alcohols, withaniol, C25H33O4OH and somnirol, C32H43O6OH, a new dihydric alcohol, somnitol, C33H44O5(OH)2, an acidic hydrolytic product, withanic acid, C29H45O6 COOH, a nitrogen containing component, C12H16N2, phytosterol, C27H46O and ipuranol, C25H38O2 (OH)2. In addition a mixture of fatty acids, consisting of stearic, cerotic, palmitic, oleic and linoleic acids; an essential oil and sugar were obtained.

Alkaloidal compounds

Later on Majumdar (1952, 1955), examined the roots of Indian variety from Bengal and South African varieties and identified several nitrogenous bases and partially characterized seven amorphous bases namely withanine, withananine, withananinine, pseudo-withanine, somniferine, somniferinine, somnine along with nicotine as eight component. The first six compounds were found to be alkaloids and the seventh one is a disintegrated product of withanine. Among these, withanine was found to be the main alkaloid, with 38% of the total alkaloid content. Schwarting et al. (1963) made the major breakthrough by isolating and characterizing eight bases present in the extract namely, tropine, pseudotropine, 3α-tigloyloxytropane, choline, cuscohygrine, dl-isopelletierine, anaferine and anahygrine the latter two being the new ones (Schwarting et al. 1963). Further, Schröter et al. (1966) isolated a pyrazole alkaloid withasomnine from the root of W. somnifera.

Jayaprakasam et al. (2004) purified novel withanamides A-I from the methanolic extract of W. somnifera fruits. The structure of these compounds was determined by using serotonin, glucose and long-chain hydroxyl fatty acid moieties (Jayaprakasam et al. 2004).

Withanolides

After reports of alcoholic compounds by Power and Salway (1911) and alkaloids by Majumdar (1952, 1955), Lavie and co-workers in series of papers reported a new group of steroidal lactones characterized by C28 basic skeleton with 9 C atoms side chain and a 6 membered lactone ring from W. somnifera which they termed as “Withanolides” (Lavie et al. 1965, 1966, 1968). The withanolides possess a highly oxygenated cholestane type side chain bearing an extra methyl group at C-24. viz. withaferin A (Lavie et al. 1965; Kirson et al. 1970). Table 1 describes various withanolides identified in different chemotypes of W. somnifera with their molecular formula, IUPAC name, physiochemical analysis and geographical location whereas Fig. 1 provides structures of major withanolides.

Table 1 Bioactivity and other parameters of withanolides isolated from W. somnifera from different geographical locations
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Fig. 1
figure 1figure 1

Major withanolides isolated from Withania somnifera

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Withaferin A is a polyfunctional steroid-lactone. The structure of withaferin A and its 2,3-dihydro derivative has been elucidated by chemical studies and X-ray crystallography by Lavie et al. (1965, 1966) and Kirson et al. (1970). Basic skeleton of withaferin A was confirmed after several selenium dehydrogenations which lead to isolation of a derivative of cyclopentenophenantharene and of a trimethylnaphthalene. It is mainly known for its anti-cancerous property. It possesses three likely positions which might be involved in in vivo alkylation reaction with the biological nucleophiles thus result in activity. These includes position 3 in ring A, the epoxide i.e. position 5 (or 6) and position 24 in unsaturated lactone ring E. Anti-tumor activity of the analogues with cholesterol side chain was weaker than that of withanolides thus proved the presence of unsaturated lactone in the side chain is necessary for the activity (Yoshida et al. 1979). There are number of withanolides which has structure similar to withaferin A i.e. unsaturation at position 2 and 24 and epoxy group at position 5 and 6 (Fig. 1). Withanolide D which has been isolated in year 1968 by Lavie et al. has the same structure similar to withaferin A but presence of hydroxyl group at position 20 was found instead of position 27 (Lavie et al. 1968).

Withanolide A which was previously isolated from Withania coagulans has been isolated from roots of W. somnifera in 1971 (Menssen and Stapel 1973).

A chlorinated withanolides i.e. withanolide C was isolated from chemotype III of W. somnifera (Besselle and Lavie 1992). Structure of withanolide C revealed the opening of 5β,6β epoxide ring and possesses chloro group at position 5.

Glotter et al. (1973) isolated nine withanolides (withanolide E–M) and found five withanolides viz. G, H, I, J, K possess an unusual Δ8(14) double bond. Very few reports are available on the presence of Δ8(14) double bond in natural steroids. Withanolide F, possesses a double bond instead of 5β,6β epoxy group present in withanolide E (Glotter et al. 1973). X-ray analysis of withanolide E and F has disclosed that the side-chain possesses the unusual 17α-orientation (Lavie et al. 1972). Withanolide S was obtained during a study of biogenesis of withanolides in W. somnifera involving combination of various types through cross pollination. In NMR, it has revealed a close similarity to withanolide E, the only difference was lack of epoxide ring signal at δ 3.20 and the presence of secondry axial hydroxyl group at δ 4.10 (Glotter et al. 1977).

In the AB ring system, 2,5-dien-1-one, present inter alia in withanolide F, G, G2, H, J, L, M, N, O, P, Q, U. Withanolide Q has the same NMR signal in the low-field region for three vinylic proton as found in withanolide G thus allowing assignment of 2,5-dien-1-one structure to the AB ring system. Withanolide Q and R both have a hydroxyl group at position 23 whereas comparison of NMR signals confirms that the only difference between the side chain is the absence of hydroxyl group at position 27 in the latter.

Structure of withanolide Y has been elucidated as 5α, 6α-epoxy-7α, 17α, 20R-trihydroxy-1-oxo-22R-witha-2, 24-dienolide by X-ray single structure analysis. Among all the withanolides it is the only example of having hydroxy group at 7th position (Abraham et al. 1975).

Kirson et al. (1971) investigated W. somnifera growing in North-Western India and have reported the isolation of eight new steroidal lactones. e.g. withanone and tubacapsenolide F along with six derivatives. Upfield chemical shift of H-2 and H-3 at δ 6.07 and δ 6.56 respectively, suggest the presence of 2-en-1-one withasteroid moiety having epoxy group at 6, 7 position which can be further verify by presence selectively of 2-mercaptoethanol with the 5β,6β-epoxy steroids substituting the epoxide by a six-membered oxyethylene-2′-thio ring whereas it failed to show such reactivity on 6α,7α-epoxy withasteroids as in withanone (Misra et al. 2008). Tubacapsenolide F has structure similarity with withaferin A, the only difference is the presence of hydroxyl group at position 17 instead of position 27.

Velde et al. isolated a naturally occurring steroidal lactone of withanolide G i.e. dunawithagenin (Velde and Lavie 1981) and Δ16-withanolide (Velde and Lavie 1982) has been isolated from the W. somnifera Chemotype III. Dunawithagenin was found to be naturally occurring steroidal lactone of withanolide G and has hydroxyl group at position 1, 3 and 20 whereas Δ16-withanolide is considered to be an intermediate in the biosynthesis of withanolide E. It was the first withanolide having unusual α-oriented side chain in its structure.

Rahman et al. in 1991 isolated withasomniferin-A and in 1992 isolated sominolide and sominone (Rahman et al. 1992) from W. somnifera. Withasomniferin-A has epoxy group at position 6, 7 as in withanone but the major difference is the presence unsaturation in ring A at position 4. Also, it has only one hydroxyl group at position 17 but interestingly sominolide has 14,15-epoxy group, inspite of 4,5 or 6,7 epoxy group as present in withaferin A and withanone respectively. Sominone has been characterized by the absence of epoxy group but it has three hydroxyl group at position 1, 3 and 27.

Withasomidienone has also been isolated by Rahman and coworkers and spectroscopic studies showed the presence of three double bond at position 1, 4 and 24 instead of position 2 and 24 as in most of withanolides (Rahman et al. 1993; Misra et al. 2005).

Choudhary et al. (1996) isolated withaoxylactone and somnifericin from W. somnifera. Withaoxylactone is the only withanolide which has been characterized by the presence of two epoxy moiety, one is at 5, 6 position and another is at 14, 15 position whereas somnifericin is differentiated by the presence of four hydroxy group at position 4, 5, 6 and 27.

Ali et al. (1997) isolated five new withanolides from the stem bark of W. somnifera, collected from the southern region of Delhi, namely withasomnilide, withasomniferanolide, somniferanolide, somniferawithanolide and somniwithanolide. Withasomnilide is structurally similar to withanone but differ only in the position of hydroxyl group as in withasomnilide hydroxyl group present at position 5, 8 instead of 5 and 17 as in withanone whereas somniferanolide has been elucidated as epoxy group at position 16, 17, two hydroxyl group at position 8 and 11 and three double bond at position 2, 5, and 24. Furthermore, withasomniferanolide, somniferawithanolide and somniwithanolide has been characterized by the absence of epoxy group in basic moiety.

Anjaneyulu and Rao (1997) isolated three new withanolides, withasomniferol A, withasomniferol B and withasomniferol C from the non-basic fraction of the benzene and ethyl acetate extracts of the roots of W. somnifera. Withasomniferol A has the same basic moiety as present in withanone, the only difference is the presence of hydroxyl groups. Structural elucidation of withasomniferol A showed the presence of hydroxyl group at position 5, 20 and 27 instead of position 5 and 17 as in withanone whereas withasomniferol B has hydroxyl group at position 5 and 20 and also having only one unsaturated position i.e. 2 instead of 2 and 24 in withanone.

Abou-Douth (2002) isolated 4-deoxywithaperuvin. This has been characterized by the presence of five hydroxyl group at position 5, 6, 14, 17 and 20. Jayaprakasam and Nair (2003) isolated viscosalactone B from W. Somnifera. Viscosalactone B is structurally analog to withaferin A as presence of 6,7 epoxy group but it has only one double bond at position 24 instead of 2 and 24 as in withaferin A and also has three hydroxyl group at position 3, 4 and 27 (Jayaprakasam et al. 2003).

A number of withanolide having glycocidal linkage and withanolide derivatives has also been isolated from W. somnifera summarized in Tables 2 and 3.

Table 2 Bioactivity and other parameters of steroidal glycosides isolated from Withania somnifera
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Table 3 Bioactivity and other parameters of withanolides derivatives isolated from Withania somnifera
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Miscellaneous compounds

Misra et al. 2012 has isolated two compounds, 2,5-dioxo-3tetratriacont-3’-enyl-1,4-dioxane and 1,4-dioxo-3,25,26 trihydroxyergosta-24(28)-ene from W. somnifera.

Chemotypes

Extensive studies carried out by Israeli group led by Lavie et al. revealed presence of various chemotypes in W. somnifera. Their study revealed that Withania has fairly wide geographic distribution and several chemotypes have been identified based on the type and content of various substituted steroidal lactones of withanolide series present in the species. In Israel several chemotypes of W. somnifera have been identified differing in their total leaf content of withanolides with various substitution patterns (Lavie et al. 1972; Glotter et al. 1973; Kirson and Glotter 1980). These substitutions are characteristic of each chemotype and seem to be genetic in character. Three chemotypes (I, II, III) of W. somnifera L. (Dun.) were found to occur in Israel (Abraham et al. 1968). Figure 2 describes the three chemotypes of W. somnifera with their major withanolides as reported by Ganzera et al. (2003), Kirson et al. (1977), Leyon and Kuttan (2004), Ray and Jha (2001), Sethi and Subramanian (2006) and Shohat et al. (1978).

Fig. 2
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Major chemotypes reported from Withania somnifera

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The main withanolides of chemotype I and chemotype II is withaferin A and withanolide D respectively (Kirson and Glotter 1980). In chemotype III, two groups of compounds have been characterized, one with compounds possessing a normal stereochemistry at C17 (i.e. β-oriented side chain) e.g. Withanolide G and J, and other with α-oriented side chain e.g. withanolide E and F (Lavie et al. 1972). Distinct ecotypes of W. somnifera L. Chemotype III have been found to grow in Israel, which are characterized by possessing the same major components in the plant but differing in the relative concentrations. The existence of such ecotypes was confirmed by semi-quantitative reversed phase high performance liquid chromatography (RP-HPLC).

Chemotype I Chemotype I grow in southern and central parts of Israel. The main character in the chemistry of this plant is its ability to introduce OH groups at various sites of the carbon skeleton. The predominant features of this plant are substituents found in A/B rings, the 4β-OH, 5,6-β-epoxy system and absence of OH group at C-20 of the side chain (Kirson and Glotter 1980). Withaferin A was the major product obtained from the leaves of chemotypes-I (ca. 0.2% of the dried leaves). Chemotype I has also been reported from India (Kaushik et al. communicated).

Chemotype II This chemotype of W. somnifera occurs mainly in the northern parts of Israel. Withanolide D was found to be present in major quantities (0.53 g/kg dry leaves) others being found in quantities of mg. The compounds present in chemotype II are mainly characterized by presence of OH group at C-20, 4β-OH and 5,6β-epoxy system. Withanolide G being an exception to this was supposed to be as unreacted precursor of this group (Choudhary et al. 2004).

Chemotype III Located in southern coastal plains of Israel. This type of W. somnifera contains two groups of compounds all characterized by presence of OH group at C-20 position. Some of them possess three separate double bonds in A, B, and C rings. One type of compound contains a α-oriented side chain e.g. withanolide E and F and other containing a normal β-oriented side chain e.g. withanolide G and J. Withanolide E and J also contain C17–OH group (Choudhary et al. 2004).

Israeli chemotype

Three chemotypes of W. somnifera (L.) Dun., Solanaceae, each containing different steroidal lactones of the withanolide type, have been found to occur in Israel; they have been called types I, II and III (Abraham et al. 1975; Gupta et al. 2011). Morphological differences could not be detected between the three types, although each of them has a definite and separate area of distribution. No qualitative ontogenetic changes in the withanolide content could be observed.

Italian chemotypes

In Italy, W. somnifera is only present in Sicily and Sardinia. Absence of substitution at C-4, configurations 20S and 14α-OH, and predominance of 17α-OH compounds in Sicilian species is evident of the fact that Sicilian plants belong to the Israelian chemotype III. The only difference is in the presence of withanolide J as chief constituent as compared to the withanolide E in Israelian species. Similar research studies on Sardinian plants led to the isolation of identical components as of Sicilian, thus confirming the Italian chemical race (Nittala and Lavie 1981).

Indian chemotypes

The chemotype I growing in India is characterized by presence of two main constituents i.e. withaferin A and withanone (Besselle and Lavie 1987). A number of samples of leaves from North Western India were found to show the predominant characteristics of chemotypes I. Beside this, intermediate of chemotype I and chemotype II were also discovered in the study. This chemotype is characterized by the presence of withaferin A and withanolide D as its chief constituents (TERI 2006).

Hybridisation of various chemotypes

Many hybrids of various chemotypes of W. somnifera have been attempted, which lead to production of new withanolides. The withanolides presents in hybrid of various chemotypes are shown in Fig. 3 and details of such new recombination are discussed below.

Fig. 3
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Major withanolides presents in hybrid of various chemotypes of W. somnifera

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Hybrids of chemotype II (Israel) and Indian I (Delhi)

Hybrid plants of W. somnifera from cross-pollinations of chemotypes II (Israel) and Indian I (Delhi) have been examined. The chief constituent isolated was 14β-hydroxywithanone (6α, 7α-Epoxy-5α, 14β, 17α-trihydroxy-1-oxo-22R-witha-2, 24-dienolide) This compound is the first example of a 14β-substitution among withanolides (Nittala and Lavie 1981).

Hybrids of chemotype III (Israel) and Indian I (Delhi)

Hybrid plants of W. somnifera obtained from cross-pollinations of chemotypes III (Israel) and Indian I (Delhi) have also been found to contain 14β-hydroxywithanone as major compound similar to hybrids of chemotype II (Israel) and Indian I (Delhi). Beside this, three new compounds viz.14α-hydroxywithanone(5α,14α,17α-trihydroxy-6α,7α-epoxy-1-oxo-22-R-witha-2,24 dienolide), 6β,7β-epoxywithanone (5α,14α,17α-trihydroxy-6β,7β-epoxy-1-oxo-22 R-witha-2,24-dienolide) and 2,4,6-trien-1-one (14α, 20α-dihydroxy-1-oxo-22 R-witha-2,4,6,24-tetraenolide) have been reported (Nittala and Lavie 1981).

Hybrids of chemotype II (Israel) and South African Chemotype

Crossbreeding of W. somnifera chemotype II (Israel) by South African chemotype led to the isolation of six compounds, three of them being new ones. The principle withanolide was found to be withanolide D followed by 24,25-dihydro-4-dehydrowithanolide D, 24,25-dihydrowithanolide D, 4-dehydrowithanolide D, 2,3-dihydrowithanolide D and withaferin A (Sethi and Subramanian 1976).

Pharmacological utility

The biological activity of Ashwagandha is mainly attributed to steroidal components, withanolides present in them, making plant useful in a wide variety of pathological states. Various activity reported by various workers is discussed in this section of review:

Adaptogenic activity

Withanolide 5β, 6β-epoxy-1-oxo-witha-2-ene-27-ethoxy-olide isolated from the roots of W. somnifera when evaluated for the stress-related parameters, namely serum lactate dehydrogenase (LDH) activity, serum creatine phosphokinase (CPK) activity, serum corticosterone levels, and serum lipid peroxidation (LPO) level showed significant decrease in a serum CPK, LDH, and LPO levels was observed in animal pretreated with 5β, 6β-epoxy-1-oxo-witha-2-ene-27-ethoxy-olide at the dose of 2.5 mg/kg body weight) in comparison to control when subjected to C-H-R stress. Thus concluded, the withanolide, 5β, 6β-epoxy-1-oxo-witha-2-ene-27-ethoxy-olide could prove to be an effective agent to counteract C-H-R stress (Misra et al. 2008).

In another study, the adaptogenic activity of a standardized extract of WS roots was also investigated against a rat model of chronic stress (CS) and significant hyperglycaemia, glucose intolerance, increase in plasma corticosterone levels, gastric ulcerations, male sexual dysfunction, cognitive deficits, immunosuppression and mental depression were induced and these perturbations were attenuated by W. somnifera at a dose of 25 and 50 mg/kg (p. o) in comparison to 100 mg/kg (p. o), of dose of prostaglandin administered 1 h before footshock for 21 days (Bhattacharya and Muruganandam 2003). Results indicate that W. somnifera, like Prostaglandin, has significant antistress adaptogenic activity confirming the clinical use of the plant in Ayurveda. Similar result was further confirmed by another investigation in which ethanolic extract of roots of W. somnifera 23 mg/kg (p.o), on acute stress induced biochemical and immunological perturbations in mice improved the swim duration in mice and significantly restored back the stress induced alterations in plasma cortisol, blood glucose and triglyceride levels (Anju 2011). Very recently, Candelario and his group proved that differential activation of GABA receptor subtypes explains a potential mechanism for its reported adaptogenic properties (Candelario et al. 2015).

Anti-inflammatory activity

The transcription factor NFκB and the signaling pathways that regulate its activation play a critical role in normal and pathophysiological immune responses thus leaf extract of W. somnifera and its major constituent withaferin A, was tested for its effect on NFκB. It was found it potentially inhibits NF κB activation by preventing the tumor necrosis factor-induced activation of IκB kinase β whereas other W. somnifera derived steroidal lactones, such as withanolide A is far less effective (Kaileh et al. 2007). Additionally, withaferin A hampers NFκ-β activation by targeting cysteine 179 located in catalytic site of IKK-β (Heyninck et al. 2014). Thus concluded that pure withaferin A or withaferin A-enriched W. somnifera extract have a considerable NFκB inhibitors activity, which hold promise as novel anti-inflammatory agents for treatment of various inflammatory disorders and/or cancer.

Methanolic fractions of the plant extract possess anti-inflammatory activity comparable to that of a 5 mg/kg dose of hydrocortisone sodium succinate and were attributed to the high content of biologically active steroids (withanolide) in the plant of which withaferin A is the major component (Al-Hindawi et al. 1992). Similarly, hydro-alcoholic extract of W. somnifera also possessed marked anti-inflammatory effect against denaturation of protein in vitro. The effect was plausibly due to the alkaloid and withanolide contents of W. somnifera (Chandra et al. 2012). The anti-inflammatory activity of the plant was further supported by a study conducted by Khan et al. (2011) in assessment of cholinesterase and lipoxygenase inhibitors activity of the plant. In a very recent study leaf water extract and one of its active chloroform fraction was found to suppress the proliferation of activated microglia by causing cell cycle arrest at Go/G1 and G2/M phase along with decrease in cell cycle regulatory protein expression such as PCNA and Cyclin D1. Both the extracts attenuated the TNF-α, IL-1β, IL-6, RNS, and ROS production via downregulating the expression of inflammatory proteins like NFkB and AP1 and also restricted the migration of activated microglia by downregulating metalloproteinase expression and may prove to be a potential therapeutic candidate for the suppression of neuroinflammation in the treatment of neurodegenerative diseases (Gupta and Kaur 2016).

Anti-tumour

As anti-inflammatory, cardioactive and central nervous system effects of W. somnifera involve angiogenic processes thus it was hypothesized that the W. somnifera extracts might contain angiogenesis inhibitors. It was found that withaferin A inhibited cell proliferation in human umbilical vein endothelial cell (HUVECs) with IC50 value of 12 nM through a process associated with inhibition of cyclin D1 expression (Mohan et al. 2004). Withaferin A was found to have anti-angiogenic activity in vivo at doses that are 500-fold lower than those previously reported to exert anti-tumor activity in vivo thus hold a promise for potent anti-tumor drug. Studies at molecular level revealed that withaferin A inhibits binding of Sp1 transcription factor to VEGF (vascular endothelial cell growth factor) gene promoter, in order to exert its antiangiogenic activity (Prasanna et al. 2009). These results clearly indicate the antiangiogenic potential of withaferin A in modulating antitumor activity. Withanolides from W. somnifera inhibited growth of central nervous system, lung, human breast and colon cancer cell lines comparable to doxorubicin. Withaferin A inhibited growth of breast and colon cancer cell lines more effectively than doxorubicin. These results suggest W. somnifera extracts may prevent or inhibit tumor growth in cancer patients and suggest a potential for development of new chemotherapeutic agents. Withaferin A, sitoindoside IX, 4-(1-hydroxy-2, 2-dimethylcyclpropanone)-2, 3-dihydrowithaferin A, 2, 3-dihydrowithaferin A, 24,25-dihydro-27-desoxywithaferin A, physagulin D (1 → 6)-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranoside, 27-O-β-d-glucopyranosylphysagulin D, physagulin D, withanoside IV, 27-O-β-d-glucopyranosylviscosalactone B, 4, 16-dihydroxy-5β, 6β-epoxyphysagulin D, viscosalactone B from the leaves of this species and diacetylwithaferin A were tested for their antiproliferative activity on NCI-H460 (Lung), HCT-116 (Colon), SF-268 (Central Nervous System; CNS and MCF-7 (Breast) human tumor cell lines. Withaferin A and its derivatives exhibited IC50 ranging from 0.24 ± 0.01 to 11.6 ± 1.9 μg/mL. Viscosalactone B showed IC50 ranging from 0.32 ± 0.05 to 0.47 ± 0.15 μg/mL whereas its 27-O-glucoside derivative exhibited IC50 between 7.9 ± 2.9 and 17.3 ± 3.9 μg/mL (Jayaprakasham et al. 2003b).

Withanolides inhibit cyclooxygenase enzymes, lipid peroxidation, and proliferation of tumor cells because several genes that regulate cellular proliferation, carcinogenesis, metastasis and inflammation are regulated by activation of nuclear factor-κB (NF-κB) (Ichikawa et al. 2006). Thus withanolides was supposed to suppress NF-κB activation. Suppression was not cell type specific, as both inducible and constitutive NF-κB activation was blocked by withanolides. Overall, it is suggested that withanolides inhibit activation of NF-κB and thus NF-κB regulated gene expression, which may explain the ability of withanolides to enhance apoptosis and inhibit invasion and osteoclastogenesis. Beside this, withanolide D, an important bioactive withanolide, purified from the leaves of an Indian chemotype (NMITLI 135) exhibited antileukemic activity, targeting multiple pathways along with ceramide accumulation through N-SMase 2 activation, ultimately inducing apoptosis in neoplastic cells (Mondal et al. 2012). In an another investigation worked on the structure activity relationship analysis of withanolides with respect to its anti-proliferative activity against an array of cell lines, human head and neck squamous cell carcinomas cell lines, breast cancer cell line and non-malignant human cell line confirmed the importance of the presence of a Δ 2-1-oxo-functionality in ring A, 5β,6β-epoxy or 5α-chloro-6β-hydroxy grouping in ring B, and nine-carbon side chain with a lactone moiety for cytotoxic activity whereas the presence of –OH or –OR groups at C-4, 7, 11, 12, 14, 15, 16, 17, 18, 19, 20, 23, 24, 27, and 28 were not contributors to the observed antiproliferative activity (Zhang et al. 2012).

Anti-oxidant activity

Clinical effectiveness of antioxidants generally showing that reactive oxygen species (ROS) and oxidative damage are important factors in the processes involved. Withanamides A, B, C, D, E, F, G, H and I (alkaloids from W. somnifera) and three withanolides from the methanolic extract of W. somnifera fruits were tested for their ability to inhibit lipid peroxidation in a model system using large unilamellar vesicles. All the nine withanamides inhibited lipid peroxidation at 1 and at 0.5 μg/mL and one withanolide inhibited the lipid peroxidation by 82% at 10 μg/mL. Commercial antioxidants, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ) were also tested in this assay at 1 μg/mL and showed 80, 81 and 85% of inhibition, respectively (Jayaprakasam et al. 2004). Thus results suggest that hydroxylated long-chain acyl group may be responsible for the potent antioxidant activity exhibited by novel withanamides. Other compounds viz. Sitoindosides VII-X and withaferin A were found to increase the free-radical scavenging enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) levels in the rat brain frontal cortex and striatum (Bhattacharya et al. 1997). An increase in these enzymes would represent increased antioxidant activity and a protective effect on neuronal tissue.

Immunomodulatory activity and hematopoiesis

The role of W. somnifera as immunomodulator has been extensively studied. Sitoindoside IX and sitoindoside X, isolated from W. somnifera Dun, have immunomodulatory and CNS effects (anti-stress, memory and learning) in doses of 100–400 g/mouse and produced statistically significant mobilization and activation of peritoneal macrophages, phagocytosis and increased activity of the lysosomal enzymes secreted by the activated macrophages. Thus W. somnifera attenuate cerebral function deficits in the geriatric population and to provide non-specific host defence (Rahman et al. 1999). Root extract of W. somnifera has immunomodulatory effects in three myelosuppression models in mice: cyclophosphamide, azathioprin, or prednisolone and significant increases in hemoglobin concentration, red blood cell count, white blood cell count, platelet count, and body weight were observed in W. somnifera treated mice compared to untreated control mice (Ziauddin et al. 1996). The authors also reported significant increases in hemolytic antibody responses toward human erythrocytes, which indicated immunostimulatory activity.

Treatment with W. somnifera root extract at the dose of 20 mg/dose/animal; i.p. was found to enhance the total WBC count (17,125 cells/mm3) on 10th day, bone marrow cellularity (27 × 106 cells/femur) as well as α-esterase positive cell number (1800/4000 cells) that was found to increase significantly (P < 0.001) after the administration of Withania extract (Davis and Kuttan 2000). Antibody titre and the number of plaque forming cells (PFC) in the spleen were also found to increase with Withania extract treatment along with the antigen (SRBC). Maximum number of PFC (985 PFC/106 spleen cells) was obtained on the fourth day. Delayed type hypersentivity reaction in mice (Mantoux test) was also inhibited. Administration of Withania extract also showed an enhancement in phagocytic activity of peritoneal macrophages (76.5 pigmented cells/200) when compared to control (31.5/200 cells) in mice. These results confirm the immunomodulatory activity of W. somnifera extract, which is a known immunomodulator in indigenous medicine. The immunologic effects of W. somnifera on four types of immune cells in a human sample were investigated. After 96 h, significant increases in the expression of CD4 on CD3+ T cells and CD56+ NK cells were observed (Mikolai et al. 2009). Chandran and Patwardhan (2017) revealed the immunomodulation mechanism of W. somnifera involved five bioactive viz. 2,3-dihydrowithaferin A-3-β-O-sulfate, β-sitosterol, daucosterol, withaferin-A, and withasomniferol-A which are capable of regulating 15 immune system pathways through a network of six bioactive-targets interactions and ten other protein-proteins interactions (Chandran and Patwardhan 2017).

Neuritic regeneration activity

Neurodegenerative diseases are characterized by progressive dysfunction and death of cells that frequently affect specific neural systems, implying some form of selective vulnerability. In order to reconstruct neuronal networks, neuritic regeneration and synaptic reconstruction must take place in the damaged brain thus the compounds that would facilitate the regeneration of neurites and the reconstruction of synapses, even in severely damaged neurons, provides new insights for drug development to prevent, treat, and cure these diseases. Withanolide A (10 mmol kg/1 day/1, for 13 days, p.o.) could regenerate neurites and reconstruct synapses in severely damaged neurons and also recovered Ab (25–35)-induced memory deficit in mice (Kuboyama et al. 2005). Some withanolides from methanolic extract of the roots of W. somnifera showed significant neurite outgrowth activity at a concentration of 1 mM on a human neuroblastoma SH-SY5Y cell line (Jayaprakasam et al. 2003). Possible mechanism of neuroprotective action of the root extract of W. somnifera Dunal (WS) is inhibition of nitric oxide production, which is known to mediate neurodegeneration during stress. Treatment of adult mice with W. somnifera extract for 30 days during stress significantly reversed the stress induced NADPH-d activation via suppressing corticosterone release and activating cholineacetyltransferase, which in turn increase serotonin level in hippocampus to inhibit NADPH-d (Bhatnagar et al. 2009). Thus down regulation of nNOS and neurochemical alterations of specific neurotransmitter systems attributed to neuroprotective action of W. somnifera.

Anxiety and depression

W. somnifera has been used to stabilize mood in patients with behavioral disturbances. Bioactive glycowithanolides (WSG), isolated from W. somnifera roots, have been assessed at the dose of 20 and 50 mg/kg, orally once daily for 5 days for anxiolytic and antidepressant actions in rats. It was found that WSG gives results compared to standard bendodiazepine lorazepam in the dose of 0.5 mg/kg, ip for anxiolytic studies and standard tricyclic anti-depressant, imipramine in the dose of 10 mg/kg, ip for the antidepressant investigations (Bhattacharya et al. 2000b). In an another study, W. somnifera in the dose of 100, 200 or 500 mg/kg, oral dependently increased the time spent and entries into the open arms on EPM test and showed the anxiolytic activity. It also helped to potentiate the anxiolytic action of diazepam (0.5, 1 or 2 mg/kg, ip) at subeffective dose i.e. 50 mg/kg, oral (Gupta and Rana 2007). Similar result were reported by Kaurav et al. 2012, revealed aqueous and methanolic extracts W. somnifera (50 mg/kg) successively decreased the marble burying behavior activity without affecting motor activity which is comparable to the activity of standard fluoxetine, ritanserin and parachlorophenylalanine (Kaurav et al. 2012).

Nootropic effect

W. somnifera is a traditional Ayurvedic medicine, used for centuries as a memory-enhancing agent. The plant, plant extract and isolated withanolides (the major active principles) have been extensively investigated in several laboratories for their neuropharmacological effects and a number of reports are available confirming their nootropic action. W. somnifera root extract in the dose of 50, 100 and 200 mg/kg; orally improved retention in a step-down paradigm in mice also in the dose of 50, 100 and 200 mg/kg; orally reversed the scopolamine (0.3 mg/kg)-induced disruption of acquisition and retention and attenuated the amnesia produced by acute treatment with electroconvulsive shock (ECS), immediately after training (Dhuley 2001). Alzheimer’s disease is a syndrome induced by ibotenic acid (IA) lesioning of the nucleus basalis magnocellularis and thus caused a marked cognitive deficit. Equimolar amounts of sitoindosides VII-X and withaferin A in the dose of 20–50 mg/kg significantly reversed both IA-induced cognitive deficit and the reduction in cholinergic markers after 2 weeks of treatment (Bhattacharya et al. 1995).

Antigenotoxic effect

Pretreatment with Withaferin A in Syrian golden hamsters significantly reduced the frequency of micronucleated polychromatic erythrocytes (MnPCEs) and chromosomal aberrations such as chromosomal break, gap, minute, and fragment caused by 7, 12-dimethylbenz (a) anthracene (DMBA). Results thus demonstrated the antigenotoxic effect of withaferin-A in DMBA-induced genotoxicity in the bone marrow of golden Syrian hamsters (Panjamurthy et al. 2008).

Antihepatotoxic

Glycowithanolides, consisting of equimolar concentrations of sitoindosides VII-X and withaferin A, isolated from the roots of W. somnifera on 10 days of oral administration in graded dose of 10, 20 and 50 mg/kg attenuate iron overload (FeSo4, 30 mg/kg, i.p.) induced hepatotoxicity in rats in comparison to Silymarin in the dose of 20 mg/kg, p.o. and that may be due the antioxidant action of glycowithanolides WSG (Bhattacharya et al. 2000a).

Influence of W. somnifera root powder on the levels of circulatory ammonia, urea, lipid peroxidation products such as TBARS (thiobarbituric acid and reactive substances), HP (hydroperoxides) and liver marker enzymes such as AST (aspartate transaminase), ALT (alanine transaminase) and ALP (alkaline phosphatase), for its hepatoprotective effect in ammonium chloride induced hyperammonemia were investigated. W. somnifera offers hepatoprotection by influencing the levels of lipid peroxidation products and liver markers in experimental hyperammonemia and this could be due to (1) the presence of alkaloids, withanolids and flavonoids, (2) normalizing the levels of urea and urea related compounds, (3) its free radical scavenging property and (4) its antioxidant property (Harikrishnan et al. 2008). Sabina et al. (2013) investigated the protective effect of W. somnifera against paracetamol-induced hepatotoxicity and found that treatment with W. somnifera significantly reversed elevated levels of liver marker enzymes and bilirubin. It also helped to improve the total protein content, histological observations and antioxidant status which was affected by paracetamol treatment (Sabina et al. 2013).

Antimicrobial

Monomeric glycoprotein with a molecular mass of 28 kDa isolated from the W. somnifera root tubers demonstrated potent antimicrobial activity against the phytopathogenic fungi and bacteria tested and exerts a fungistastic effect against Aspergillus flavus, Fusarium oxysporum, Fusarium verticilloides and antibacterial activity against Clvibacter michiganensis subsp. michiganensis by inhibiting spore germination and hyphal growth in the tested fungi (Girish et al. 2006). In an another study conducted using both disc diffusion and serial dilution method different extracts of leaf, flower, root and stem part of W. somnifera inhibited six bacteria (two Gram +ve and four Gram −ve bacteria) i.e. Staphylococcus aureus (Gram +ve), Bacillus Subtilis (Gram +ve), Escherichia coli (Gram −ve), Raoultella planticola (Gram −ve), Pseudomonas aeruginosa (Gram −ve), Enterobactor aerogens (Gram −ve), and two fungi Candida albicans and Aspergillus flavus to varying degrees whereas water extract of leaves of W. somnifera showed highest activity against R. planticola (Singariya et al. 2011, 2012a, b, Datta et al. 2011). Beside antibacterial and antifungal activity, hydro-alcoholic extract of W. somnifera roots also showed the inhibition of bursal disease virus at maximum 99.9% in its highest nontoxic concentration, 25 μg/mL in cytopathic effect reduction assay (Pant et al. 2012).

Antidote activity

A large family of peptides includes proteins of different origin, neurotoxins, myotoxins, cardiotoxins/cytotoxins and enzymatic toxins are the peptides present in snake venom (Girish et al. 2004). A glycoprotein WSG isolated from W. somnifera is an inhibitor of hyaluronidase of Naja naja and Daboia russelii venom and IC50 value was found to be 52 and 36 μg for N. naja and D. russelii venoms, respectively. It also inhibits phospholipase A2 of the toxic cobra venom, thus it may help in preventing the rapid diffusion of toxins (Machiah et al. 2006). Recently Kumar et al. (2015a) also reported the antidote activity of W. somnifera against arsenic induced toxicity.

Anti-Parkinson’s activity

Parkinson’s disease (PD) is a neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra pars compacta. To assess the efficacy of W. somnifera extract 100 mg/kg was feed to the mouse. The midbrain and corpus striatum of parkinson affected mouse showed increased levels of catalase, superoxide dismutase and malondialdehyde; and reduced levels of glutathione and glutathione peroxidase compared to the normal mouse while treatment with Withania extract 100 mg/kg for 7 days significantly improved all these enzyme levels compared to Withania untreated Parkinson affected mouse brain thus suggest that Withania is a potential drug in treating Parkinson affected oxidative damage (RajaSankar et al. 2009).

Cardio protective effect

Wistar rats were used to evaluate the cardio protective mechanisms of W. somnifera, in the setting of ischemia and reperfusion (IR) injury (Mohanty et al. 2008). Post-ischemic reperfusion injury resulted in significant cardiac necrosis, apoptosis, and decline in antioxidant status and elevation in lipid peroxidation in the IR control group as compared to sham. W. somnifera prior-treatment favorably restored the myocardial oxidant-antioxidant balance, exerted marked anti-apoptotic effects [upregulated Bcl-2 (p < 0.001) protein and attenuated TUNEL positivity (p < 0.01)], and reduced myocardial damage as evidenced by histopathologic evaluation.

Anti-convulsant

Kulkarni et al. 2008, analyzed the effects of W. somnifera extract (100 or 200 mg/kg, po) against pentylenetetrazol (PTZ) seizure threshold in mice. The drug was tested alone and in combination with exogenous gamma-amino butyric acid (GABA), a GABA receptor agonist or with diazepam. W. somnifera increased the PTZ seizure threshold for the onset of tonic extension phase. Co-administration of a sub-effective dose of W. somnifera (50 mg/kg, po) with a sub-protective dose of either GABA (25 mg/kg, ip) or diazepam (0.5 mg/kg, ip) increased the seizure threshold.

Male infertility

Stress has been reported to be a causative factor for male infertility. W. somnifera has been documented in Ayurveda and Unani medicine system for its stress-combating properties. Treatment of infertile male with W. somnifera extract is found to inhibit lipid peroxidation and protein carbonyl content and improved sperm count and motility and also men recovered the seminal plasma levels of antioxidant enzymes and vitamins A, C, and E and corrected fructose (Ahmad et al. 2010). The treatment effectively reduced oxidative stress, as assessed by decreased levels of various oxidants and improved level of diverse antioxidants. Moreover, the levels of T, LH, FSH and PRL, good indicators of semen quality, were also reversed in infertile subjects. Mahdi et al. 2011 also defined the role of stress in male infertility after measuring various biochemical and stress parameters before and after treatment. Study reported that root powder at the dose of 5 g/day for 3 months treat stress-related infertility improved the level of anti-oxidants and improved overall semen quality in a significant number of individuals (Mahdi et al. 2011; Shukla et al. 2011). Kumar et al. (2015b) revealed that W. somnifera also reversed the effect of sodium arsenite administration on sperm counts and sperm motility and also maintains the cellular integrity of testicular cells leading to normal functioning of it.

Activity in agriculture

Herbicidal activity

Aqueous, methanol and n-hexane shoot and root extracts of 5, 10, 15 and 20% w/v (fresh weight basis) concentrations of W. somnifera were tested against the germination and seedling growth of Parthenium in which aqueous and methanol extracts markedly suppressed the germination, root and shoot growth of Parthenium (Javaid et al. 2011). The activity was checked by two bioassay method, first is foliar spray bioassay, the aqueous and methanol shoot extracts of 10% w/v (on a dry weight basis) concentration were sprayed on 1-week and 2-week-old pot-grown parthenium seedlings. Two subsequent sprays were carried out 5 and 10 days after the first spray. The aqueous and methanol extracts significantly reduced the length and biomass of parthenium shoots.

In another soil amendment bioassay, the crushed shoots of W. somnifera were incorporated in the soil at 1–5% w/w. Parthenium seeds were sown one week after the residue incorporation and plants were harvested 40 days after sowing. All the soil amendment treatments significantly reduced seed germination by 43–89%.

Pesticidal activity

Spodoptera litura is a Noctuid moth which is considered as an agricultural pest. Treatment of sixth instar larvae and pupae of the polyphagous pest Spodoptera litura with an acetone extract of leaves of W. somnifera, caused toxicity, molt disturbances, formation of larval-pupal, pupal-adult intermediates and adultoids. Thus W. somnifera acts as an insect growth regulator causing disruption of the endocrine mechanism regulating molting and metamorphosis (Gaur and Kumar 2010).

Conclusion

The literature survey revealed that W. somnifera is an important source for the group of compounds, withanolides, which are having important pharmacological activity. Beside this, preliminary studies have also found that group of constituents of Withania exhibit a variety of therapeutic activity. It is concluded from the review that there is significant variation in the biological activity of the withanolide studied, which may be due to stereochemical specificity in their steroidal structure. The extensive survey of literature revealed that W. somnifera is an important source of many other pharmacologically and medicinally important chemicals, sitoindosides and various useful alkaloids.

The plant has also been widely studied for their various pharmacological activities like immunomodulatory activity and hematopoiesis, adaptogen, antivenom, anti-inflammatory, antitumor properties. Various other effects like immunomodulation, antioxidant, anxiolytic, hypolipidemic, antibacterial have also been studied.

Studies of total withanolide content in the hybrid plants and their respective parents also revealed that a hybrid plant contains more total content of withanolides as compare to parent plant. Also chemotype variation of W. somnifera has not been studied much in India, so this review throws a fresh perspective of chemotypic study of this plant in India, production of superior hybrids with potential for the commercial exploitation and development of improved varieties with distinct chemoprofiles targeting specific bioactive molecules for different pharmacological applications.

Also, information on the different chemotypes is not available for Withania plants found in different parts of world. Such studies would be interesting to develop genetic linkages.

Although the results from this review promises new prospects for the use of Withania somnifera as a multi-purpose medicinal agent, several limitations currently exist in the recent literature.