Concurrent analysis of bioactive triterpenes oleanolic acid and ß-amyrin in antioxidant active fractions of Hibiscus calyphyllus, Hibiscus deflersii and Hibiscus micranthus grown in Saudi Arabia by applying validated HPTLC method.

In this study, we developed a validated HPTLC method for concurrent analysis of two natural antioxidant triterpenes, oleanolic acid (OA) and ß-amyrin (BA) in the biologically active fractions (petroleum ether, toluene, chloroform, ethyl acetate and n-butanol) of aerial parts of three Hibiscus species (H. calyphyllus, H. deflersii and H. micranthus). The chromatography was conducted on normal HPTLC (ready to use glass-plate coated with silica gel 60 F254) plate with chloroform and methanol (97:3, V/V) used as mobile phase. The derivatization of the developed plate was done with p-anisaldehyde and scanned at λmax = 575 nm. Well resolved and intense peaks of OA and BA were obtained at Rf = 0.36 and 0.57, respectively. The linear regression equation/correlation coefficient (r2) for OA and BA were Y = 6.65x + 553.35/0.994 and Y = 9.177x + 637.23/0.998, respectively in the linearity range of 100-1200 ng/spot indicated good linear relationship. The low values of %RSD for intra-day/inter-day precision of OA (1.45-1.61/1.38-1.59) and BA (1.52-1.57/1.50-1.53) suggested that the method was precise. The recovery/RSD (%) values for OA and BA were found to be 99.21-99.62/1.39-1.95 and 98.75-99.70/1.56-1.80, respectively assures the reasonably good accuracy of the proposed method. Fifteen samples were analyzed to check the content of OA and BA by using the developed HPTLC methods. The content of OA in different samples were found to be 3.87 (HmP) > 1.212 (HcP) > 0.673 (HdC) > 0.493 (HdP) > 0.168 (HdE) > 0.059 (HcC) > 0.015 (HcE) > 0.008 (HmT) µg/mg of the dried weight of extract. However the content of BA was found as: 2.293 (HmP) > 1.852 (HdT) > 0.345 (HdC) > 0.172 (HmT) > 0.041 (HdE) > 0.008 (HcC) µg/mg of the dried weight of extract. Some Hibiscus species fractions exhibited good antioxidant potential like: HcE (IC50 = 17.6 ±â€¯1.8) > HdB (IC50 = 32.16 ±â€¯0.9) > HmP (IC50 = 80.4 ±â€¯4.5) > HmT (IC50 = 99.7 ±â€¯8.2) when compared with ascorbic acid (IC50 = 14.2 ±â€¯0.5), while other fractions exhibited only mild antioxidant capability. The developed HPTLC method can be further exploited for analysis of these markers in the quality assessment of raw material as well as herbal formulations available in the market.

Anticancer activity and concurrent analysis of ursolic acid, ß-sitosterol and lupeol in three different Hibiscus species (aerial parts) by validated HPTLC method.

The genus Hibiscus contains about 275 species of flowering plants widely grown in the tropics and sub-tropics. The available literature revealed that several Hibiscus species exhibited excellent anticancer activity against several cancer cells like lung, breast, and liver. This motivated the authors to explore the anticancer property of other Hibiscus species (Hibiscus calyphyllus, H. deflersii and H. micranthus) along with development of a validated HPTLC method for the concurrent analysis of three anticancer biomarkers (ursolic acid, ß-sitosterol and lupeol) in different Hibiscus species. The anticancer activity of various fractions (petroleum ether, toluene, dichloromethane, ethyl acetate and n-butanol) of all the Hibiscus species (aerial parts) were evaluated in vitro against HepG2 and MCF-7 cell lines using MTT assay. The HPTLC analysis was carried out using chloroform and methanol as mobile phase (97:3; v/v) on 20 × 10 cm glass-backed silica gel 60F254 plates and analyzed different phytoconstituents present in all fractions at λ = 575 nm wavelength. Of the tested fractions of H. calyphyllus, H. deflersii and H. micranthus, HdP (H. deflersii petroleum ether fraction) exhibited the most potent cytotoxic effect on HepG2 and MCF-7 (IC50: 14.4 and 11.1 µg/mL, respectively) cell lines. Using the developed HPTLC method a compact and intense peak of ursolic acid, ß-sitosterol and lupeol were obtained at Rf = 0.22, 0.39 and 0.51, respectively. The LOD/LOQ (ng) for ursolic acid, ß-sitosterol and lupeol were found as 42.30/128.20, 13.20/40.01 and 31.57/95.68, respectively in the linearity range 100-1200 ng/spot. The obtained result showed maximum presence of ursolic acid, ß-sitosterol and lupeol (5.50, 11.85 and 7.47 µg/mg, respectively) in HdP which also supported its strong anticancer effect. Our data suggest that H. deflersii petroleum ether fraction (HdP) can be further subjected to the isolation of active cytotoxic phytoconstituents and establishment of their mechanism of action. The maiden developed HPTLC method for concurrent analysis of anticancer biomarkers may be further employed in the in process quality control of herbal formulation containing the said biomarkers.

An integrated metabolome and transcriptome analysis of the Hibiscus syriacus L. petals reveal the molecular mechanisms of anthocyanin accumulation.

Hibiscus syriacus L. var. Shigyoku is a new double-flowered bluish-purple variety in China that changes color during flower development from bluish-purple to light purple. There is limited information on the anthocyanin accumulation patterns and associated transcriptome signatures in Shigyoku from D1 (bud) to open flower (D3). Here, we employed a combined transcriptome and metabolome approach to understanding the mechanism of this color change. Our results demonstrate that cyanidins, pelargonidins, delphinidins, petunidins, peonidins, and malvidins were differentially accumulated in Shigyoku petals. The anthocyanin biosynthesis started in D1, was significantly upregulated in D2 (semi-open flower), and reduced in D3. However, malvidins, pelargonidins, and peonidins could be associated with the bluish-purple coloration on D2. Their reduced accumulation in D3 imparted the light purple coloration to Shigyoku petals on D3. Significant contributions in the color change could be associated with the expression changes in anthocyanin biosynthesis genes i.e., LARs, ANSs, DFRs, UGT79B1, C3'Hs, 3ATs, and BZ1s. The UFGTs were associated with the higher accumulation of glycosylated anthocyanins in D2 and D3. Furthermore, the changes in the expressions of the MYB and bHLH transcription factors were consistent with the anthocyanin accumulation. Finally, we discussed the possible roles of Jasmonic acid, auxin, and gibberellic acid signaling in regulating the MBW complex. Taken together, we conclude that H. syriacus petal coloration is associated with anthocyanin biosynthesis genes, the MBW complex, and phytohormone signaling.