ESTABLISHMENT OF QUALITY PARAMETERS OF Quisqualis indica LEAVES THROUGH SOPHISTICATED ANALYTICAL TECHNIQUES

. Quisqualis indica ( Q. indica ; Rangoon creeper) is found in Asia and finds its place in Ayurvedic texts, ethno-medicine as well as modern research. Its leaves contain important constituents like rutin, quisqualis acid, trigonelline, L-proline and L-asparagine. Traditionally, the leaves are used as antipyretic, anti-flatulent, anti-inflammatory, anti-septic, and anti-diarrhoeal. Modern pharmacological research also supports these claims. However, this plant remains unexplored phytochemically, which restricts any means for standardization of its formulations. The present research focuses on analysis of leaves of Q. indica using sophisticated chromatographic and spectral techniques. Thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), gas chromatography-mass spectrometry (GC-MS) techniques were used. After several pilots, TLC analyses, an HPTLC fingerprint of methanolic extract of the leaves was performed using chloroform: methanol: ethyl acetate (7: 3: 3) solvent system, which showed 12 peaks at 254 nm and 9 peaks at 366 nm. GC-MS analysis of the methanolic extract detected 7 known phytochemicals, some of them having pharmacological importance. This research may serve the parameters for quality control of Q. indica leaves in herbal industries, in the detection of adulteration of its formulations as well as open new avenues for phytochemical research, including isolation of a marker compound.


INTRODUCTION
Quisqualis indica (Q. indica, Figure 1) is a ligneous vine that belongs to the family Combretaceae. In this plant, the leaves are opposite or elliptical. In India, it is grown as an ornamental plant, while it is distributed across the world in tropical countries, especially in China, the Philippines, Bangladesh, Myanmar, and Malaysia [1][2][3]. It is commonly known as Rangoon creeper or Chinese Honeysuckle (English). Local names of Q. indica include Madhumalati (Hindi), Modhumalati (Bengali), Parijat (Manipuri), Vilayati Chambeli (Marathi), Radha Manoharam (Telugu), Niyogniyogan (Filipino), Quiscual (Spanish) and Shih-chun-tzu (Chinese) [4][5]. Each plant contains several phytochemicals in its various parts showing different pharmacological activities and toxicities, likewise Q. indica Linn. also shows many pharmacological activities due to the presence of medicinally active compounds.
Other parts of Q. indica also have significant ethnomedicinal uses. Fruits are tonic, and anthelmintic, used in nephritis and gargling, diarrhea, and as astringent. Seeds are used in diarrhea as an antiseptic, febrifuge for high fevers, vermifuge, and anthelmintic. Roots are used in rheumatism, diarrhea, and as anthelmintic [9][10][11].
This research study may aid in the identification and characterization of phytoconstituents by chromatographic fingerprints obtained using sophisticated HPTLC and GC-MS techniques. Using the reports, one can check adulteration and facilitate standardization of herbal formulations containing leaves of Q. indica. It will also promote further research studies and the isolation of phytochemicals for the betterment of the community.

Chemicals and reagents
Methanol was purchased from the Rankeem Chem Trade Enterprise (purity >99%, analytical AR grade). Chloroform, ethyl acetate, ammonia and formic acid were obtained as gift sample from the Molychem Laboratory.

Collection and sampling
Five year old, mature fresh leaves of Q. indica were collected from the Medicinal Garden of RK University (Latitude 22.24006, Longitude 70.90098), Rajkot -India in the monsoon, August 2014, and compared with standard literature for authentication. The leaves were ovate in shape, 5-13 cm  2-5 cm in size, pale green in colour with coarse surface texture. A herbarium [SOP/COG/398/2014] was submitted to repository at School of Pharmacy, RK University, and certified by the Botanist from School of Science, RK University.

Extraction
After collection, the leaves were dried in a hot air oven at 50 o C. Leaves of Q. indica were powdered and 25.0 g dry powder was extracted with 100 mL methanol for 24 hours by maceration at room temperature. The macerated solution was filtered and the filtrate was allowed to dry at 50 o C. The obtained solid mass was stored for further experiments.

Pilot TLC and HPTLC studies
For the best resolution in HPTLC study, the mobile phase system was developed on TLC. Methanolic extract of leaves was used to develop TLC. Chloroform, methanol, ethyl acetate, ammonia and formic acid were used in the development of TLCs. After several trials in TLC studies, the best mobile phase system was identified. The proportions of the solvents were modified for several TLC plates to obtain precise and clear spots in fingerprinting. Chloroform: methanol: ethyl acetate (7: 3: 3) system gave best separation in TLC and forwarded for HPTLC analysis.
HPTLC analysis for fingerprinting of methanolic extract was carried out at the Department of Chemistry, Saurashtra University with chloroform: methanol: ethyl acetate (7: 3: 3) system. The HPTLC fingerprinting was obtained on HPTLC plates containing silica gel C60 F254 as stationary phase, manufactured by E. Merck KgaA. CAMAG Linomat 5 was used for sample application. Peak height and area were selected for evaluation, while the measurement was done using the principle of absorption. The sample was dissolved in Methanol. CAMAG TLC Scanner 3 was used for scanning the plates in daylight as well as at 254 nm and 366 nm.

GC-MS studies
Qualitative GC-MS analysis of the sample named Madhumalati, containing methanolic extract of Q. indica leaves was performed using GC-MS at the Department of Chemistry, School of Science, RK University -INDIA. Agilent 5977B MS occupied to 7820A GC was used to analyze. HP-5 capillary column (30 m × 0.32 mm; 0.25 µm film thickness) was used for GC-MS studies [12][13][14][15][16]. NIST library of the GC-MS was used to identify the detected compounds.

Pilot TLC and HPTLC studies
The mobile phase chloroform: methanol: ethyl acetate (7: 3: 3) showed distinct spots in TLC. Hence, it was further used for HPTLC fingerprinting. HPTLC plates were scanned at 254 nm and 366 nm as shown in Figure 2 and their densitometric spectra are as shown in Figure 3.

GC-MS studies
From GC-MS analysis of the methanolic extract of Q. indica leaves, 7 phytochemicals as shown in Table 3 were identified using NIST library. Chromatogram of the GC analysis is shown in Figure 4, while spectra from the MS analysis is shown in Figure 5.    The phytochemicals detected from the GC-MS analysis of the methanolic extract of Q. indica leaves were reported with significant biological activities. Decylundecyl ester carbonic acid is used as an acidifier as well as to inhibit the production of uric acid. 3,7,11,15-tetramethyl-2hexadecen-1-ol is useful for providing oligosaccharides to the body. However, others did not possess known pharmacological activities.
It is significant to note that Agarwal et al. [17] reported 15, 12, and 18 compounds qualitatively via GC-MS analysis, in methanol, ethyl acetate, and hexane extracts respectively [17]. In addition to that, Sutar et al. [18] investigated the phytochemical and biological activities of Q. indica leaves and evaluation of secondary metabolites and characterization of isolated compounds was done by TLC, GC-MS analysis, NMR, and FTIR [18]. Potphode et al. had performed bioethanol production of Q. indica leaves by GC-MS analysis [19].

CONCLUSION
This exclusive research work may open up further ideas to facilitate the standardization of herbal formulations containing Q. indica leaves. The obtained chromatographic fingerprints may aid to check adulteration and in the quality control of herbal formulations. This research study can also be used in further research for the isolation of compounds or as a marker for other processes.