Biomedical Analysis on Phytopharmaceuticals

Abstract

In view of importance of biomedical analysis of phytopharmaceuticals present investigations / studies have been taken into consideration to evaluate and report the biological activities of three plants species commonly available in Pakistan. The biomedical analysis / procedure comprises of different techniques which ultimately helps to ascertain and establish the uses and application of phytopharmaceuticals as a therapeutic agent on the basis of results. Based on these guidelines and research activities (i.e., bioactivity results) alternative system of medicine supports treatment and prophylaxis of a number of disease to a vast number of people worldwide. One of such system is very popularly known as ―Herbal System of Medicine‖, while the products of this system of medicine are classified as ―Herbal Medicine‖. The herbal medicines are widely used to prevent and treat diseases either in the form of crude or standardized extracts on the basis of the presence of one or two very promising pure compounds established through biochemical analysis. Phytopharmaceuticals have made great contribution in promoting and establishing human health. Recent studies have indicated their significant role in the development of new drugs, commonly classified as phytomedicines. These phytomedicines have great therapeutic potential and a number of prominent molecules are under phase II and III clinical trials. Present studies were also designed to conduct biomedical analysis on three plant species with the aim to explore their biological activities and to authenticate their earlier usage and application with scientific justification as well as to see the possibility of using in some new indications. The medicinal plants include; Adenanthera pavonina, Peltophorum pterocarpum and Piper nigrum while the biological activities include; anti-oxidant, anti-bacterial, anti- inflammatory / analgesic, neuropharmcological and hepatoprotactive activities along with acute toxicity assessments using hexane and ethanol extracts and three pure compounds; β- Sitosterol glucoside, Bergenin and Piperine consequently, the dissertation has been divided into below mentioned five parts.  Part I: This has two chapters.  Chapter one deals with the role of phytopharmaceuticals derived from various sources, information about production in different phytogeographical regions xxiii of Pakistan, medicinal market (size and value) and therapeutic usage and application.  Chapter two covers the botanical description of selected plants (Adenanthera pavonina, Peltophorum pterocarpum and Piper nigrum)along with their traditional and modern medicinal uses based on literature survey reports biological and pharmacological activities.  Part II: Describes the methods adopted for pharmacognostic analysis. This included extraction, screening of phytoconstituents, isolation and identification of pure compounds β-sitosterol glucoside, bergenin and piperine from leaves of Adenanthera pavonina, Peltophorum pterocarpum and fruits of Piper nigrum respectively using standard test methods and biological activities include anti-oxidant, anti-bacterial, analgesic, anti-inflammatory, neuropharmcological and hepatoprotactive assays of selected plants.  Part III: Comprises the results obtained from different biomedical analysis.  Part IV: Deals with the overall assessment and discussion on results, while  Part V: The fifth and last part consists of conclusion and suggestions. Part I: Recent research has supported to impel the precise knowledge of different plants around the world including Pakistan, to expedite the strengthening of new medicines and supplements. Scientific knowledge of the phytomedicines has started to link traditional medicines, based on pharmaceutical research and development. This has vitalize the requirement of medicinal plants as a source of therapeutic agent in modern system of medicine, and Pakistan has been reported as one of the most important country in South Asia delivering a large number of medicinal plant having therapeutic potential. Accordingly, Pakistan has been divided into four phytogeographical zones by some investigators such as Saharo-Sindian, Indian element, Irano-Turanian and Sino-Himalavian, the highest being Irano-Turanian region delivery around 45% plant species. While other investigators have divided Pakistan into five xxiv geographical region such as Hazara, Malakand, Muree Hills, Azad Kashmir and Northern areas, the highest being Hazara region which produces around 500 tons of medicinal plants annually. Chapter two of part I in the present study includes description of three plants,Adenanthera pavonina and Peltophorum pterocarpum belong to family Fabaceae while Piper nigrum belongs to family Piperaceae. The traditional uses and application have been noted quite interesting and a detailed correlation have been made in the discussion part which is based on the results of the present study as well as scientific information available elsewhere. Part II: Therapeutic potential of the test plants mainly results from the chain of phytoconstituents present in plants such as alkaloids, phenol, steroids, tannins, flavonoids, steroids, resins, and fatty acids which are capable of stimulating distinct physiological effect in body. Present work indicated that the solvents play a vital role in extraction of the phytoconstituents and isolation of pure compounds. The development of novel technologies that support prompt isolation and characterization of reputed principle molecules and these technologies should suspect the biological activity of plant extracts and pure compounds are generally results from their synergistic strength. As ethanol is distinctly polar among the other solvents therefore it extracts most and high content of bioactive phytochemicals. So preliminary screening tests may be valuable in the estimation and qualitative analysis of pharmacologically potent compounds. This is specifically true for phytochemicals that are well documented for their pharmacological activities such as alkaloids, flavonoids, tannins sterols and resins possess significant anti-nociceptive, sedative and anti-psychotic effects. Moreover saponins are well known for their ability to inhibit pain perception and anti-inflammatory as well as sedative properties and decreased spontaneous motor activity. Different classes of polyphenols like flavonoids, flavonols, flavones and tannins can exhibit anti-oxidant and hepatoprotactive activities. xxv Part III: Pharmacognostic evaluation includes detection of variousplants metabolites using ethanol extracts of all three test plants species. While isolation of pure compounds based on detection of MP, TLC, and IR analysis. The biomedical analysis includes acute systemic toxicity assessment, anti-oxidant, anti-bacterial, analgesic, anti-inflammatory, neuropharmacological and hepatoprotactive activities using standard test procedures. Results of the present study have been noted quite encouraging to use in future research studies and in authentication of early reported uses and applications. Part IV: Review of literature revealed that the plant Adenanthera pavonina has been reported to contain (β-sitosterol, β- sitosterol-3β -D- glucoside) and saponins. Earlier scientific investigation of Adenanthera pavonina showed that the crude extract possessed anti-fungal, anti-oxidant and anti-inflammatory activities. Leaves of plant aretraditionally used to treat gout and rheumatism. Review of literature showed that different parts of Peltophorum pterocarpum tree are used to treat different diseases like embrocation for pains and sores. The traditional healers use the leaves in the form of decoction for treating skin disorders. Stem infusion and flowers of Peltophorum pterocarpum is used in muscular pain. A large number of chemical constituents have been reported from different parts of the tree and these chemical constituents are known to exhibit some important biological activities such as anti-microbial and anti-oxidant activities. Many therapeutic activities of Piper nigrum attributed to the presence of major pungent alkaloid piperine apart from other chemical constituents. It is widely used in different traditional systems of medicine like Ayurvedic and Unanii System of medicines as rheumatoid arthritis.Piperine exhibits diverse pharmacological actions like anti-oxidant, analgesic, anti-inflammatory, anti-depressant, hepato-protective, anti-bacterial activities. The presence of valuable phytoconstituents were detected in respective extract of selected plants (Adenanthera pavonina, Peltophorum pterocarpum andPiper nigrum) include saponins, alkaloids, carbohydrates, cardiac glycosides, tannins, flavonoids, steroids, and resins.A pale yellow amorphous solid of β-sitosterol glucoside was obtained from ethanol xxvi extract of Adenanthera pavonina leaves. White crystals of bergenin were isolated from methanol extract of Peltophorum pterocarpum leaves, while yellow needle shaped crystals of piperine were delivered from ethanol extract of Piper nigrum fruits. It is crucial to conduct toxicity study for safety and efficacy of phytopharmaceuticals / herbal products. The estimation of LD50 and ED50 values obtained from the present study showed that test samples that administered intra peritoneal (in mice) and oral (in rats) at different tested dose level were safe and nontoxic. Invitro anti-oxidant analysis usually provides a reliable method to evaluate phytopharmaceuticals to authenticate their usage and application to cure inflammatory conditions. In the present study the tested plants were found to be magnificent in anti-oxidant property and can act as a reducing agent because of the hydroxyl group present in major phytochemicals. Results of anti-oxidant analysis indicated that the piperine possessed highest reducing power (1.039) along with high percentage of reducing power capacity (72.1%). Pure isolated compounds β-sitosterol glucoside from Adenanthera pavonina leaves, bergenin from Peltophorum pterocarpum leaves and piperine from Piper nigrum fruits showed significant anti-oxidant effect among all tested samples in a concentration dependent manner but less than standard drug ascorbic acid. The anti-bacterial activity of test samples of three selected plants were evaluated against Gram positive and Gram negative bacterial strains. Test extracts and pure compounds had shown enhanced activity against both groups of bacterial strains indicating broad spectrum activity which may play an important role in the management of bacterial infection by decreasing expression of bacterial genes or inhibiting bacterial growth and reducing the production of bacterial toxins. Piperine isolated from Piper nigrum fruits possessed highest zone of inhibition (20 mm) against Gram negative bacteria Acinetobacter baumannii. Minimum inhibitory concentration (0.195%) showed by ethanol extract of Piper nigrum fruits and pure compound bergenin of Peltophorum pterocarpumleaves against Gram negative bacteria Acinetobacter baumannii and Klebsiella pneumoniae respectively. Time to kill Escherichia coli by piperine started to produce action at 60 minutes that was lasted for long duration (1440 minutes) with log cfu/ml 0.2. While pure compound bergenin of Peltophorum pterocarpum leaves was started its bactericidal activity at 120 minutes that was lasted till 240 minutes and showed strong log cfu/ml 0.03 but for short period of time. xxvii The anti-nociceptive and anti-inflammatory studies were under taken to evaluate and validate the analgesic and anti-inflammatory potential of selected plants species against different pain models. Test samples demonstrated analgesic activity in dose dependent manner and indicated both central and peripheral analgesic mechanism. This may due to higher concentration of polyphenols and flavonoids present. These compounds mediate anti-oxidant activity by activating anti-oxidant enzymes like SOD (super oxide dismutase), CAT (catalase), GP (glutathione peroxidase) and responsible for free radical scavenging activity, as free radicals are involved in pain stimulation. Anti-nociceptive activity of isolated compounds, hexane and ethanol extracts of Adenanthera pavonina and Peltophorum pterocarpumleaves demonstrated in dose dependent manner,piperine and ethanol extract from Piper nigrumfruits exhibited its analgesic effect at moderate doses (10 mg/kg body weight) both centrally and peripherally. Among all tested samples, β-sitosterol glucoside an isolated pure compound of Adenanthera pavonina leaves was found more effective against pain induced by thermal method (tail immersion assay) as compared to hexane extract of Peltophorum pterocarpum leaves and pure compound piperine isolated from Piper nigrumfruits. According to the results of second thermal method (hot plate assay) analgesic activity of piperine, an isolated pure compound from Piper nigrum fruits was found stronger than ethanol extract of Peltophorum pterocarpum leaves and hexane extract of Adenanthera pavoninaleaves. Analgesic strength of test samples were evaluated by mechanical method (pain induced by analgesy meter) and the results were demonstrated in a particular order β- sitosterol glucoside, was observed most effective than bergenin an isolated pure compound of Peltophorum pterocarpum leaves and ethanol extract of Piper nigrumfruits. Significant reductions in writhes induced by acetic acid (chemical method) were presented by Peltophorum pterocarpum. The inflammatory effect induced by carrageenan could be associated with free radical attack on plasma membrane. Free radical, prostaglandin and nitric oxide will be released when administrating with Carrageenan for 1to5 hours. The response of test samples in suppression of edema induced by carrageenan was noted in second phase of inflammation in dose dependent mode. This possible mechanism could be due to inhibition of free radicals generated like nitric oxide, hydroxyl radicals and superoxide anions showing the anti- histamine property. xxviii Among all tested plants anti-inflammatory evaluation of ethanol extract ofPiper nigrum showed highest suppression of edema (0.149, 97.450%) induced by carrageenan in second phase of inflammation in dose dependent manner which confirmed the anti-histamine property of tested samples. Neuropharmcological assessment by using different behavioral test revealed that ethanol extract of Adenanthera pavonina leaves and Piper nigrum fruits as well as the hexane extract of Peltophorum pterocarpum leaves had significant anxiolytic (forced swimming test), exploratory (head dip and open field test) and neuro activator activity(stationary rod test). The estimation of hepatoprotactive effect of selected plants species showed a potential of liver protection both in prophylactic and therapeutic model in a dose dependent manner against liver injury induced by CCl4. It decreased the serum enzyme activity by induction of hepatic microsomal enzymes when compared to toxicant group. But more significant results of enzymes suppression (ALP 129.80 IU/L, ALT 31.00 IU/L, γ-GT 0.80 IU/L, DB 0.04 mg/dl and TB 0.20 mg/dl were seen in prophylactic model by bergenin at a dose of 100 mg/kg body weight. This was an effective investigation test with high negative prognostic value of liver diseases that were supplemented with histopathological examination of rat liver section. Present study also indicated the treatment of liver diseases with pure compounds of all tested plants had maximum hepatoprotactive activity at high dose in prophylactic model. This reduction indicated that pure compounds might have protected both plasma membrane and liver cells against damage thereby decreasing the leakage of serum enzymes into blood circulation may be due to anti-oxidant activity of phytochemicals, which may be helpful in controlling complication during degenerative liver diseases. Part V: The obtained data of present study suggested that Adenanthera pavonina, Peltophorum pterocarpum and Piper nigrum possess pharmacological potential to be used as therapeutic agent.These selected plants indicated a quite distinct feature from pharmacognostic evaluation and revealed comprehensive and distinct biological activities in animal’s modules. xxix However the application of these plants in some specific indication needs further study to investigate other constituents and to use standardized extracts for correlation with narrowing the differences in biological activities as well as to explore detailed pharmacological characteristics. Different pharmaceutical formulation can be prepared using these plants for further studies.