Characterization of Phyto-isolates
The compound SA-1 identified as oleanolic acid was obtained as white amorphous powder. It was acid hydrolysed and responded positively to Liebermann-Burchard test for steroids. The presence of sterol nucleus was confirmed by acid hydrolysis using 2M H2SO4. The IR spectrum showed characteristic absorption bands at (2917, 2849) for C-H stretching, and (1730) for C=O stretching thereby confirmed the presence of carbonyl group. The generation of the prominent ion peaks at m/z 457 [C30H48O3]+ was attributed to molecular ion of oleanolic acid. Other fragment ions peaks at m/z 439 [C30H47O2]+, 411 [C29H47O]+, 395 [C29H47]+, 248 [C16H24O2]+ , 235 [C15H24O2]+ supported presence of steroidal structure (Supplementary Scheme S2).
The 1H NMR spectrum of compound SA-1, methyl protons displayed one singlet at δ 0.47 for 6H assigned to H-29 & H-30 protons, two singlet at δ 0.4 & 0.45 assigned to H-23 & H-24 protons, and three singlet at δ 0.52, 0.78, 0.81 were assigned to H25, H-26, & H-27 protons respectively. There was a characteristic triplet signal at δ 5.21 with J value of 15.12 assigned to H-12. The protons of OH and COOH group displayed characteristic broad signal at δ 12.42 & 12.6 respectively. Remaining methylene and methine unit protons displayed broad multiplet signals between δ0.47 to 2.13. The 13C NMR spectrum data of SA-1, exhibited important signals at δ 14.21 for C-23 and C- 24, signal at δ 24.71 for C-29 & C-30. The signal at δ 29.06 was assigned to C-26 & C-27. A signal at δ 22.71 was assigned to C-25. The signal at δ 32.02 for C-31, signal at 178 assigned to carbonyl carbon. The two characteristic signals at δ 125.77 and 127.28 were assigned to C12 and C13 respectively. The carbonyl group signal appeared at δ 178.45. The remaining 19 carbons displayed their signals from δ 14.1 to 77.02 (Table 1).
The HMBC correlations of H-C(3) with C(1), C(2), C(4), C(23), and C(24), of H-C(9) with C(5), C(8), and C(10), of H-C(12) with C(11), C(13), C(14), and C(18), of H-C(18) with C(13), C(14), C(16), and C(28), and of H-C(22) with C(28), indicated the relative positions of these groups in the molecule SA-1 (Figure. 3a).
The IR data, Mass fragmentation pattern, 1H NMR and 13C NMR chemical shifts of the isolated compound SA-1 were comparable with related compound viz, oleanolic acid. On the basis of spectral data analysis and acid hydrolysis based chemical identification, the structure of isolated compound SA-1, was finally elucidated as oleanolic acid.
The compound SA-4 identified as 3-hydroxylup-12-(13)-ene-17-carboxylic acid, was obtained as yellow amorphous powder. It is obtained chemically by acid hydrolysis. It responded positively to Liebermann-Burchard test for steroids and formed effervescence with sodium bicarbonate solution indicating the presence of carboxylic acid group in the molecule. The chemical identification was done by acid hydrolysis method using 2M H2SO4 which confirmed the presence of sterol nucleus. The IR spectrum showed characteristic absorption bands for (2917, 2848) for C-H stretching, and (1735) for C=O stretch to confirm the presence of carbonyl group. The +ve ion mass spectrum showed molecular ion peak at m/z 456 [C30H48O3] + corresponding to M+ ion, and 455 [C30H47O3]+ corresponds to M-1 peak, which indicated saturated nature of molecule. The characteristic signals at m/z 439 [C30H47O2] +, 411 [C29H47O] +, 395 [C29H47]+, and 235 [C15H24O2]+ suggested presence of steroidal structure (Supplementary scheme S3).
The 1H-NMR spectrum of compound SA-4, methyl protons displayed one singlet at δ 0.65 for 6H assigned to H-29 & H-30, two singlet at δ 0.81& 0.85 assigned to H-22 & H-23 proton respectively, and one singlet at δ 0.95 was assigned to H24, H-25, & H-26 protons. There was a characteristic triplet signal at δ 5.20 with J value of 15.1 Hz assigned to H-12. The protons of hydroxy and COOH group displayed characteristic broad signal at δ 12.49 & 12.56 respectively. Remaining methylene and methine unit protons displayed broad multiplet signals at δ 0.95-3.72. The 13C NMR spectrum data of SA-4, exhibited important signals for two methyl carbons at δ 17.97 C-22, C-23, C-29 & C-30. The signal at δ 27.2 was assigned to C-26 & C-27. A signal at 18.01 was assigned to C-25. The signal δ 178 assigned to carbonyl carbon. The two characteristic signals at δ 125.42 and 127.35 were assigned to C12 & C13 respectively. The remaining 19 carbons displayed their signals from δ 14.1 to 72.53 (Table 2).
The HMBC correlations of H-C(3) with C(1), C(2), C(4), C(23), and C(24), of H-C(9) with C(5), C(8), and C(10), of H-C(12) with C(11), C(13), C(14), and C(18), of H-C(18) with C(13), C(14), C(16), C(19) and C(27), and of H-C(21) with C(27), indicated the relative positions of these groups in the molecule SA-4 (Figure 3b).
The IR, data, Mass fragmentation pattern, 1H and 13C NMR chemical shifts of the isolated compound SA-4 were comparable with related compound viz, Hydroxylupic acid. On the basis of spectral data analysis and acid hydrolysis based chemical identification, the structure of isolated compound SA-4, was finally elucidated as 3-hydroxylup-12-(13)-ene-17-carboxylic acid. This is a new compound reported for the first time in S. alata species. The spectral characterization of molecular structure of isolated compounds SA-1 and SA-4 was confirmed and supported using standard literature (19–22).
In-vitro COX-1 and COX-2 inhibitory assay
COX-1 and COX-2 catalyze the biosynthesis of prostaglandin H2 from the arachidonic acid substrate. The inhibition of COX-1 results in some undesirable side-effects, whereas COX-2 inhibition provides therapeutic effects in pain, inflammation, cancer, glaucoma, Alzheimer’s and Parkinson disease (23). COX-2, which is an inducible enzyme, COX-1 is constitutive, that is, present even in the absence of inflammatory conditions. In addition to the pro-inflammatory prostaglandins, COX-1 is responsible for the synthesis of those prostaglandins that are necessary for maintaining the integrity of gastro-intestinal mucosa. A higher inhibition of COX-1 increases the tendency of a drug to induce gastric ulcers and related complications (24).
The IC50 values of indomethacin for COX-1 and COX-2 were observed as 53.00 µM and 36.56 µM respectively. The results of the in vitro COX-1 and COX-2 inhibitory studies revealed that the isolated compounds (SA-1 and SA-4 potentially inhibit COX-2 (IC50 = 61.68-87.25 µM range) over COX-1 104-128 µM range). Whereas, isolated compound SA-4 (COX-1/COX-2 = 104/61.68) were found to be potent inhibitor of COX-2 than SA-1 (COX-1/COX-2= 128.4 / 87.25) (Figure 4). To understand the inhibitory activity of these isolated compounds, they are further evaluated for their in vivo anti-inflammatory activity.
Anti-inflammatory activity
Anti-inflammatory activity of the phytocompounds (SA-1 and SA-4) was evaluated as per the method of Winter et al with minor modifications. Carageenan was used to induced paw Edema in Wistar albino rat. Carrageenan induced paw edema is a standard assay for acute inflammation that is effectively employed to evaluated the phytoisolate against anti-inflammatory activity (25). Edema is produced by a sequential release of inflammatory mediators such as histamine, serotonin, kinnins, prostaglandins and bradykinins which leads to fluid accumulation (26). Edema is characteristic of acute inflammatory response (27). The release of histamine or serotonin occurs in the first phase (Upto 1 hr) and the second phase (over 1 hr) is associated with the production of bradykinins (28). It is well known that the third phase of the edema induced by carrageenan, in which the edema reaches its highest volume, is characterized by the presence of prostaglandins and other compounds of slow reaction (29,30).
Indeed, previous studies have shown that this compound play a pharmacological role in inflammation (31). The finding of the present study revealed that the phytoisolates SA-1 and SA-4 shows dose dependent anti-inflammatory activity by suppressing the rat paw edema.
Ulcerogenic activity
It has been reported for that non-steroidal anti-inflammatory agents are inadequately dissolvable in gastric acid and stay in contact with the stomach wall for a more extended period, thus producing a highly dangerous local concentration. This leads to local irritation of stomach wall followed by ulceration. This prompts to local irritation of the stomach wall took after by ulceration (32). Both the compounds SA-1 and SA-4 possessing in vivo anti-inflammatory activity were further screened for their ulcerogenic activity according to Cioli method. SA-4 showed significant ulcerogenic potential compared to the control treated group at a dose of 12 and 24 mg/kg. A strong correlation between the potency of NSAID’s as inhibitor of prostaglandin synthesis and ulcerogenic activity has been observed (33).
Most potent compound SA-4 showed severity index lower than the standard drug Indomethacin. Hence, this compound may ascertain to have better safety margin on gastric mucosa than indomethacin (34).