## 3 Biochemical Profiling of _W. somifera_
### Withanolides  
Withanolides are unique classes of steroidal lactones. Over 130 withanolides are present in 15 genera of the Solanaceae family. As per some studies, withanolides are isolated from some other Leguminosae and Labiatez families, as well as from some marine organisms [5].  
_W. somnifera_ plants fabricate the largest number of withanolides comprising highly diversified functional group. Withanolides are key biochemicals, ranging from 0.001% to 0.5% dry weight concentration in the leaves and roots of _Withania somnifera_. Withanolides are a 28-carbon-containing natural steroidal lactone built on an ergostane skeleton, in which C-22 and C-26 are oxidized to form six- or five-membered lactone rings. Withanolides are also known as 22-hydroxy ergostan-26-oic acid-26, 22-lactone. Its characteristic feature is that the C8 or C9 side chains exhibit a lactone or lactol ring. However, lactone ring may be either five- or six-membered and may be fused with the carbocyclic moiety of the molecule via a C-C bond or through an O-bridge.  
Many structures are also isolated from _W. somnifera_, which are the modifications or structural variants of withanolides which cause modifications to the carbocyclic backbone or side chains, such as withaferin A, withanolide A and D, withanone. These compounds are polyoxygenated and oxidize all C atoms of the steroid nucleus [5]. A new dimeric thiowithanolide also isolated from roots from _W. somnifera_, is named ashwagandhanolide as shown in Figure 3.  
Figure 3: _Cont_.  
Withanolides act as a precursor hormone, in that it can be transformed into human physiology as needed [7]. _W. sommifera_ is also sometimes referred to as Indian Ginseng because it structurally resembles ginseng (the active constituents of _Panax ginseng_). In addition, Withanolide A also possesses anti-dementia activities, and can thus be used for neuronal regeneration.  
Withanolides Biosynthesis  
Although the formation of withanolides in _W. sommifera_ has not been fully elucidated, two pathways are reported:  
* 2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP) pathway;
* The cytosolic mevalonate (MVA) pathway;
* The plastidic1-deoxy-D-xylulose 5-phosphate [52].  
The biosynthesis starts with the formation of cholesterol by the action of acetyl coenzyme A. Acetyl-co A is combined and converted into two types of mevalonic acid; only the R-type is used in triterpene biosynthesis, the S-type being inert in nature. Following the reduction of a C-atom, the R-type is transformed into isopentenyl pyrophosphate (IPP) which, together with its isomer 3,3-dimethyl allyl pyrophosphate (DMAPP), undergoes condensation to form geranyl pyrophosphate (GPP) and, after further condensation, famesyl pyrophosphate (FPP). Two molecules of FPP condense in the presence of NADPH and squalene synthase to generate squalene which, after oxidation, forms a squalene 2,3-epoxide undergoes ring closure to form lanosterol, an intermediate of various steroidal tritrepenoid structures. The formation of 24-methylenecholesterol has still not been determined, and it is unknown whether it is a precursor of steroidal lactones or not. Nevertheless, it has been observed that hydroxylation at C-22 and the 8-lactonization between C22 and C26 of 24-methylenecholestrol are part of the Withanolide pathway as shown in Figure 4.