The Pharmacopoeial standards such as physicochemical properties are the important quality parameters of herbal drugs. Physicochemical and phytochemical characterizations are required to be carried out for establishing their identity, purity, and quality standards. Physicochemical standards were generally used for deciding the identity, purity and strength of the drug source. These parameters were also used to detect the adulterants if present in the plant material. Physicochemical properties such as total ash, acid insoluble ash, water insoluble ash, sulphated ash, water soluble extractive and alcohol soluble extractive were evaluated in all the samples of Sida species. The result is presented in Table 3. In the case of water soluble extractives, the highest extractive value was observed for aerial parts of Sida cordifolia (18.976%) and the least value was showed by root of Sida rhombifolia (5.1%). Highest alcohol soluble extractive value was noted for aerial parts of Sida cordifolia (8.712%). The Pharmacopoeial monograph of Sida cordifoila is not available in Ayurvedic Pharmacopoeia of India [1].

Qualitative test for the detection of major class of compounds is a common preliminary screening of plant-based crude drugs as it ensures the minimal phytochemical contents present in the respective extracts. Result of preliminary phytochemical screening of selected Sida species is presented in Table 4. The preliminary phytochemical analysis showed presence of phenols, flavones, alkaloids, carbohydrates and proteins as major compounds in all the selected species.

Total polyphenolics such as Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) of selected Sida species were estimated specifically for aqueous (A), ethanol (E) and hydro alcohol (HA) extracts (Table 5). TPC was calculated from calibration curve of gallic acid (R2 = 0.9991). The highest phenolic content (TPC) was observed in the hydro alcohol extract of aerial parts of Sida cordifolia. Total poly phenolics are almost comparable for all the selected species. TPC of aqueous extracts of various samples of Sida species is as follows SCR (2.352), SALR (2.0913), SAR (2.2426), SRR (2.2772), SCA (4.6216), The highest phenolic content (TPC) was observed in the hydro alcohol extract of aerial parts of Sida cordifolia (5.8121) followed by SCR (3.5479), SRR (2.6072), SAR (2.4085) and SAL (2.1095), respectively. Total polyphenolics are almost comparable for all the selected species. TPC of water extracts is lower than that of hydroalcoholic extracts. It might be due to the higher extraction capacity of water alcohol combination.

Total Flavanoid Content (TFC) of selected species was estimated for different extracts such as water, ethanol and hydroalcohol. TFC was calculated from calibration curve of Quercetin (R2 = 0.994). Highest TFC was showed by aerial parts of Sida cordifolia (4.9459) followed by roots of Sida cordifolia (3.285) in hydroalcohol extract. Aqueous extract of Sida cordifolia contains 1.485 mg equivalents of flavonoids. Sida alnifolia showed less flavonoid compared to other species.

Flavonoid/phenolics ratio was also calculated for each sample to specify the abundance of flavonoids among the total polyphenolics. Hydroalcoholic extracts of root of S. alnifolia and S. cordifolia showed highest f/p ratio (0.96 and 0.92) followed by hydroalcoholic extract of areal part of S. cordifolia (0.85), ethanolic extract of aerial part of S. cordifolia (0.84) and hydroalcoholic extract of root of S. acuta (0.82).

The occurrence of alkaloids in Sida species has chemotaxonomic relevance to Malvaceae family, since it occurs almost exclusively in Sida genus [12,13,14]. Total alkaloid content (TAC) of various Sida specis has been estimated using Bromocresol green spectrophotometric method with atropine standard. TAC was calculated from calibration curve of Atropine (R2 = 0.991). Root of S. acuta showed highest quantity of Alkaloids (9.1846) followed by roots of S. cordifolia (3.3439), S. alnifolia (1.4402), aerial parts of S. cordifolia (0.7952) and root of S. rhombifolia (0.7640). The TAC of selected species is given in Table 6.

Comparative HPTLC profiles were developed for root of various Sida species with sequential extracts such as n-hexane, chloroform and ethanol. At 254 nm, n-hexane extract (Fig. 2) showed a common band with Rf 0.50 for all the selected species. At 366 nm, compound at Rf 0.38(red), 0.45 (blue), 0.51(red) were found to be common for all the selected species. A fluorescent band at 0.17 was detected in all other species except for S. rhombifolia. On evaluating under 550 nm, common bands are observed at Rf 0.22, 0.33, 0.48, 0.63, 0.70, and 0.90. HPTLC profile of chloroform extract (Fig. 3) showed a major band at Rf 0.54 which is specific for S. cordifolia. At 366 nm, common bands are observed at Rf 0.32, 0.50, 0.67 and 0.81 for all the selected species. Under 550 nm, bands with Rf 0.40 and 0.50 are common for all the species.

HPTLC profiling of n-Hexane extracts of selected Sida species. Tracks: SCR; SALR; SAR; SRR

HPTLC profiling of chloroform extracts of selected Sida species. Tracks: SCR; SALR; SAR; SRR

At 254 nm, sequential ethanol extract showed common bands at Rf 0.17 and 0.26.Two bands at 0.08, 0.81 were observed for S. alnifolia, S. acuta and S. rhombifolia. Major bands were observed at 0.05, 0.17, 0.36, 0.62, 0.89 as common bands in all the selected species. On visualizing under 550 nm, common bands with Rf 0.07, 0.20, 0.26, 0.64, and 0.76 are observed for all the selected species.

HPTLC profile of crude ethanol extracts (Fig. 4) is almost similar in their banding pattern, and it indicates the presence of similar phytoconstituents in all the selected species. At 366 nm, major common bands were observed at Rf 0.08, 0.22, 0.40, 0.64 and 0.90. Band at Rf 0.11 and 0.83 are two additional common compounds for all other species except Sida cordifolia. A band at 0.59 was present only for S. acuta and S. rhombifolia. On evaluating under 550 nm, common bands at Rf 0.08, 0.22, 0.69, 0.77 were observed for all the selected species.

HPTLC profiling of ethanol extracts of selected Sida species

Comparative HPTLC profile of hydroalcohol extracts of selected Sida species is presented in Fig. 5, At 254 nm, common band 0.34, 0.51 was observed. A band at Rf 0.20 was observed for S. acuta and S. rhombifolia. On visualizing under 366 nm, a major band at 0.52, 0.67(blue) is found to be common for all the species. A band at 0.42 was present only for S. cordifolia, S. acuta and S. rhombifolia. At 550 nm. A band at 0.06 is absent for S. cordifolia and S. cordata. A specific band at 0.09 was observed for S. rhombifolia only.

HPTLC profiling of hydroalcohol extracts of selected Sida species. Tracks: SCR; SALR; SAR; SRR; SCOR

Chromatogram for crude water extracts were documented by HPTLC visualizer at 254 nm and 366 nm (Fig. 6). At 254 nm, major common bands were observed at 0.45, 0.6 Common bands at 0.32, 0.45 and 0.60 were observed at 366 nm for all the selected species. At 366 nm, specific bands at 0.13, 0.17 were observed for S. cordifolia. A common band at 0.32 was observed for S. acuta and S. rhombifolia. A band at 0.11 was found to be present only for S. rhombifolia. At 550 nm common bands were observed at 0.34, 0.46 and 0.52 for all the species.

HPTLC profiling of aqueous extracts of selected Sida species. Tracks: SCR; SALR; SAR; SRR

The extracts such as ethanol, water and hydroalcohol showed maximum similarity in their banding pattern. HPTLC profiling of various extracts of selected Sida species showed that most of the species except S. cordata contain almost similar type of phytochemicals. Number of matching bands for aqueous and hydro alcohol extracts shows the close similarities in their phytochemical characteristics. There are minor variations in the band intensities of separated compounds which might be due to the variation of their concentration. Variations of abundance of some compounds have been observed for the selected species. However, most of the major bands are found to be common with almost similar band intensity.

Comparative HPLC profiles were developed for the aqueous extracts of selected species. DAD chromatograms were documented at 260 nm (Fig. 7). Major compounds for S. cordifolia were observed at Rt 1.1, 1.2, 1.3, 1.8, 3.6, 4.6, 5.1, 6.6, 7.3, 9.4 and 10.5. Compounds with Rt 1.1, 1.2, 3.6, 6.6 and 9.4 are common for all the species with variation in peak area percentage. Some specific peaks were observed at 10.5, 11.1, 12.4, 13.5 and 14.9 for S. cordifolia only. Seven compounds are found to be common for S. cordifolia and S. alnifolia. The peak at 9.4 showed highest area percentage for S. alnifolia (28.76%) followed by S. rhombifolia (6.65%), S. acuta (3.7%) and S. cordifolia (1.87%).

HPLC profiling of aqueous extracts of selected Sida species. A: SCR; B: SALR; C: SAR; D: SRR