Pharmacognostic Specification and Lupinifolin Content of Derris reticulata Stem Wood

Abstract

Derris reticulata Craib is used in traditional Thai medicine for centuries. This study was carried out to investigate the pharmacognostic parameters by qualitative, quantitative analyses, cytotoxic and antioxidant activities as well as lupinifolin content of D. reticulata dried stem wood collected from 15 habitats located at various regions throughout Thailand. Macroscopic and microscopic evaluation of D. reticulata stem wood were demonstrated. For leaf measurement of D. reticulata, palisade ratio was 8.95±1.96. Upper epidermal cell area was 1173.13±56.25 µm2. Anisocytic typed stomata were found only at lower epidermis. The stomata number and stomata index in 1 mm2 were 316.53±23.03 and 20.21±1.53, respectively. Physico-chemical parameters including loss on drying, total ash, acid-insoluble ash, water soluble extractive, ethanol soluble extractive and water contents were found to be 5.7 ± 0.1, 4.6 ± 0.1, 1.0 ± 0.1, 15.5 ± 0.7, 6.8 ± 0.3 and 8.5 ± 0.3% by dry weight, respectively. Thin layer chromatographic fingerprint was also established. D. reticulata stem woods were extracted in 95% ethanol using Soxhlet apparatus. Lupinifolin in the ethanolic extracts were analyzed by thin layer chromatography (TLC) using silica gel 60 GF254 as stationary phase and hexane : ethyl acetate (6 : 4) as mobile phase. For quantitative analysis of lupinifolin, the contents were evaluated by TLC-densitometry under UV 275 nm and TLC image analysis under UV 254 nm using image J software which were respectively found to be 8.07±2.41 and 7.76±2.21 % by dry weight. The method validity of TLC-densitometry and TLC image analysis were shown that the calibration range were polynomial with 0.6 – 3 µg/spot (R2=0.9985 and R2=0.9989). The accuracy was 97.68 – 110.59 %recovery and 94.65 – 111.79 % recovery. The repeatability was 0.9-2.38%RSD and 0.74-4.18%RSD. The intermediate precision was 0.8-4.77%RSD and 0.6-3.59%RSD. LOD were 0.14 and 0.10 and LOQ were 0.41 and 0.30 µg/spot. The robustness was 2.98%RSD and 3.04%RSD, respectively. The comparison of the total lupinifolin in both methods was not significant different (p=0.11). Additionally, the ethanolic extract of D. reticulata stem wood was tested for in vitro cytotoxic activity against 5 human cancer cell lines including breast ductal carcinoma (BT-474), undifferentiated lung carcinoma (CHAGO-K1), colon adenocarcinoma (SW-620), gastric carcinoma (KATO-3), hepatocarcinoma (HEP-G2) and one human normal cell line, lung fibroblast (Wi-38). The ethanolic extract of D. reticulata stem wood exhibited no significant activity against the five cancer cell lines as well as one normal cell line with an IC50 more than 20 µg/ml. Furthermore, the free radical scavenging potentials of the ethanolic extract of D. reticulata stem wood was demonstrated with the IC50 of 907.39 µg/ml for DPPH. FRAP assay indicated that the ethanolic extract of D. reticulata stem wood had a reducing power value equivalent to 0.132 mM Fe(II)/mg extract. The total phenolic content of the ethanolic extract of D. reticulata stem wood was 44.37 ± 0.99 mg GAE/g extract. This study provided pharmacognostic specification toward fundamental standardization of D. reticulata stem wood in Thailand. Moreover, the simple TLC-densitometry and TLC with image analysis can be applied to quantitatively determine lupinifolin in this plant material.