Anti-inflammatory activity and a new compound isolated from aerial parts of Myrsine africana

A new compound was isolated from the ethyl acetate (EtOAc) fraction of the aerial parts of Myrsine africana. The compound was eluted with mobile phase of EtOAc and n-hexane at a ratio of 0.2:9.8. The structure of the compound was identified with the help of 13 C-NMR, 1 H-NMR, heteronuclear multiple bond correlation (HMBC), heteronuclear multiple quantum coherence (HMQC), nuclear overhauser effect spectroscopy (NOESY) and correlation spectroscopy (COSY). The crude methanolic extract and various fractions of the aerial parts of the plant were also screened for anti-inflammatory activity. The EtOAc and n-hexane fractions of the plant showed moderate (44.8 and 40.9%, respectively) antiinflammatory activity, while the rest of the fractions and crude extract showed low activity at concentration of 500 μg/ml against human neutrophils.


INTRODUCTION
Pakistan is endowed with the wealth of medicinal plants and has valuable heritage of herbal remedies. Like most developing countries, rural population of Pakistan still relies mainly on indigenous system of medicine for their health and health related matters (Khattak et al., 1985). Pakistan has a very rich flora due to its soil conditions and multiple ecological regions. Pakistan has about 6000 species of phanerogams. Estimates indicate that upto 700 plant species are used as medicinal and aromatic plants (Pei, 1992). According to a survey by Pakistan Forest Institute, 75 crude herbal drugs are extensively exported and more than 200 are locally traded in Pakistan. Eighty five percent of the crude herbs are *Corresponding authors. E-mail: bashirdr2001@yahoo.com, azam_bbt@yahoo.com.
Abbreviations: NMR, Nuclear magnetic resonance; TMS, tetramethylsilane; TLC, thin layer chromatography; CC, column chromatography; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum coherence spectroscopy; MHS, Modified Hank's solution; collected by the indigenous people from the forests (Rehman and Choudhary, 2003). Crude medicinal plant materials worth more than 2.3 million US $ per year, are used in Pakistan. Large quantities of crude plants are exported to the international market (6 million US $) although at low prices (Rehman and Choudhary, 2003).
This manuscript deals with isolation and characterization of the olenane type triterpene with double bond at C-9/C-11. A compound similar to this but different in stereochemistry has been synthetically reported, but this compound has been isolated for the first time from natural source, and the final structure with complete nuclear magnetic resonance (NMR) data (Allan and Spring, 1955). The screening of the plant material for biological/pharmacological activity was also done in the present work.

General experimental procedure
Jeol JMS 600 and HX 110 mass spectrometers with the data system DA 5000 was used to record HREI-MS. The 1 H-NMR spectrum was recorded in C5D5N on Bruker AMX-400 and AMX-500 NMR spectrometers with tetramethylsilane (TMS) as an internal standard using a UNIX operating system at 400 and 500 MHz, respectively. The 13 C-NMR spectrum was recorded at 100 MHz on a Bruker AMX-500 NMR spectrometer in C5D5N. The [α]D was recorded on Jasco P-2000 polarimeter. JASCO J-810 spectropolarimeter was used to record CD spectrum. Silica gel (230-270 mesh) was used for column chromatography (CC). Silica gel coated thin layer chromatography (TLC) card (GF-254, 20 × 20 cm, 0.25 mm thick, Merck) were used to check the purity of the compound and were observed under UV light (254 and 366 nm) and ceric sulphate was used as a spraying reagent.

Plant material
Aerial parts of M. africana, were collected from Hazara division, in December 2007 to January 2008. The plant was identified by Professor Dr. Habib Ahmad, Plant Taxonomist, Hazara University, Khyber Pukhtonkhwa, Pakistan.

Extraction and fractionation
The plant material was chopped into small pieces and grinded to fine powder by using an electric grinder, after shade drying. These powdered materials (7.6 kg) were then soaked (twice) in commercial grade methanol for 15 days at room temperature with occasional shaking. After 15 days, methanol soluble materials were filtered off. All the filtrates were concentrated under vacuum at 40°C using a rotary evaporator till a blackish crude methanolic extract was obtained. After extraction, the crude methanolic extract of M. africana (Cr.MA) was suspended in distilled water (400 ml) and partitioned with n-hexane (3 x 400 ml), chloroform (CHCl3) (3 x 400 ml), ethyl acetate (EtOAc) (3 x 400 ml) and butanol (BuOH) (3 x 400 ml) to yield n-hexane (50 g), CHCl3 (45 g), EtOAc (255 g), BuOH (190 g) and aqueous (210 g) fractions, as per our reported procedure . Fifty grams of Cr.MA was reserved for pharmacological/biological activities.

In vitro anti-inflammatory activity
Inflammation, a self-protective retort, induces physiological adaptations to remove pathogenic infections and reduce tissue damage (Roussin et al., 1997). Reactive oxygen species (ROS) are formed following the assemblage and activation of NADPH oxidase (phagocyte-specific enzyme). The process is initiated, during respiratory burst of non-mitochondrial oxygen (O2) uptake by NADPH oxidase system, by production of superoxide anion (O -2 ) (Tan and Berridge, 2000). A water soluble tetrazolium salt (WST-1) is used in this study for the measurement of superoxide production by neutrophils which will be activated by opsonized zymosan, inducing phagocytic activation of neutrophils. This is a more sensitive and consistent technique among other existing techniques (Costantino et al., 1998).

Isolation of human neutrophils
Human neutrophils were isolated according to the procedure of Siddiqui et al. (1995). Fresh heparinized blood was collected from healthy volunteers. Equal volume of Modified Hank's solution (MHS, pH 7.4) was added to dilute the blood and left for 20 min at room temperature. The upper layer of leukocyte was collected and layered over Ficoll paque. The pellets obtained after centrifugation were resuspended with MHS. The cells were counted using an improved Neubaur chamber. The viability of the cells was 97% measured by Trypan Blue method.

Respiratory burst assay
The activity was performed according to our reported procedure . The in vitro assay was based on the reduction of WST-1 in the presence of activated neutrophils. A total volume of 200 µl MHS (pH 7.4), containing neutrophils (1.0 -10 4 / ml), WST-1 (250 µM) and test samples was used to determine the antiinflammatory activity. Neutrophils, buffer solution and WST-1 were added to the control. The samples were equilibrated at 37°C by addition of opsonized zymosan A (15 mg/ml), prepared by mixing with human pooled serum, and the reaction was initiated. The absorbance of the reaction mixture was determined at 450 nm. For the positive control, the non-steroidal anti-inflammatory drug "indomethacin" was used which is widely used for the treatment of several inflammatory diseases. The percentage inhibitory activity showed by plant extracts was determined against blank and calculated as follows: Percentage inhibition = 100 -[(OD test compound/OD control) x 100]

Anti-inflammatory activity
The main causes of the inflammation are the immune system reaction to infection, malignancy, cellular changes, environmental agents and injury. Inflammation is a key mark in autoimmune disease. In conditions like  Hashimoto's thyroiditis, the disease process is due to inflammation, while in some other conditions like Crohn's disease, the cause of inflammation is the diseased condition (Elaine, 2006). As the herbs possess antiinflammatory characteristics, we screen crude methanolic extract and various fractions of the plant for possible antiinflammatory activity. The results obtained are shown in Table 2, which reveals that n-hexane and EtOAc fractions of the plant showed moderate (40.9 and 44.8%, respectively) anti-inflammatory activity at concentration of 500 µg/ml. The rest of the fractions and crude methanolic extract showed low anti-inflammatory activity at the same concentration.

Conclusion
The present study reveals that the EtOAc and n-hexane fractions of the plant (M. africana) showed moderate (44.8 and 40.9%) anti-inflammatory activity against human neutrophils and needs isolation of the pure constituent which possesses anti-inflammatory actions.