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Alkaloids of the Ethnic Fruit Crop Khoonphal [Haematocarpus validus (Miers.) Bakh. f. ex Forman] from Andaman and Nicobar Islands

The ‘moonseed family’ Menispermaceae consists of 75 genera and around 520 species (Watson and Dallwitz, 1992); many of the members are traditionally used for therapeutic purposes. The family contains a wide range of benzylisoquinoline alkaloids and majority of these compounds are derived from the amino acid tyrosine. Haematocarpus validus is a member of the family Menispermaceae commonly known as ‘Blood fruit’ or ‘koonphal’ used in folklore medicine by ethnic groups of Meghalaya, Mizoram, Assam, Tripura, Andaman-Nicobar Islands (India), Bangladesh, Pakistan, Indonesia, Singapore and West Java. Fruits are rich in iron content and used for treating anaemic conditions (Singh et al., 2014; Rahim et al., 2015) and for blood purification (Momin et al., 2016). The tender shoots and leaves are used for curing hepatic disorders, root extract to relieve itching and pain (Bohra et al., 2016). Even though this crop possesses immense nutritive and medicinal value, it occupies limited geographical locations and its potential uses are not yet explored. Furthermore, there are no scientific validation studies available to support the folklore knowledge on this underutilized fruit crop. In this context, it is necessary to unravel the metabolome of H. validus and the present investigation was designed to detect the nonvolatile compounds present in the methanolic International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 12 (2017) pp. 2797-2802 Journal homepage: http://www.ijcmas.com


Introduction
The 'moonseed family' Menispermaceae consists of 75 genera and around 520 species (Watson and Dallwitz, 1992); many of the members are traditionally used for therapeutic purposes. The family contains a wide range of benzylisoquinoline alkaloids and majority of these compounds are derived from the amino acid tyrosine. Haematocarpus validus is a member of the family Menispermaceae commonly known as 'Blood fruit' or 'koonphal' used in folklore medicine by ethnic groups of Meghalaya, Mizoram, Assam, Tripura, Andaman-Nicobar Islands (India), Bangladesh, Pakistan, Indonesia, Singapore and West Java. Fruits are rich in iron content and used for treating anaemic conditions (Singh et al., 2014;Rahim et al., 2015) and for blood purification (Momin et al., 2016). The tender shoots and leaves are used for curing hepatic disorders, root extract to relieve itching and pain (Bohra et al., 2016). Even though this crop possesses immense nutritive and medicinal value, it occupies limited geographical locations and its potential uses are not yet explored. Furthermore, there are no scientific validation studies available to support the folklore knowledge on this underutilized fruit crop. In this context, it is necessary to unravel the metabolome of H. validus and the present investigation was designed to detect the nonvolatile compounds present in the methanolic fractions of the leaf and fruit extracts using HR LCMS analysis. This investigation provides information on alkaloid compounds of H. validus that support the traditional knowledge on the medicinal value of this plant.

Materials and Methods
Coarsely powdered leaf and fruit samples were extracted with methanol for the detection of compounds of non-volatile nature using HR LCMS facility provided by SAIF, IIT Bombay. HR LCMS was performed using 1290 Infinity UHPLC System of Agilent Technologies, USA (1260 infinity Nano HPLC with Chipcube, 6550 iFunnel Q-TOFs). The metabolites present in the methanolic extract were identified by comparing their retention indices (RI) and mass spectra fragmentation pattern with those on the stored library available in IIT, Bombay.

Results and Discussion
The present phytochemical analysis with the methanolic extract of leaf and fruit samples of Haematocarpus validus using HR LCMS detected three true heterocyclic isoquinoline alkaloids viz., sinomenine, (S)-reticuline, ambelline, one nonheterocyclic protoalkaloid metanephrin, one tropane group alkaloid ecgonine and one quaternary ammonium compound choline. The hetrocyclic isoquinolin alkaloids viz., sinomenine, (S)reticuline, and ambelline are derived from tyrosine. The protoalkaloid metanephrin is also the part of tyrosine metabolism. Out of the six compounds detected, choline was found in both leaf and fruit samples while ecgonine and sinomenine were detected in leaves; metanephrine, (S)-reticuline and ambelline were obtained from fruits ( Table 1) The family Menispermaceae is rich in benzylisoquinoline alkaloids and many of them are used as medicinal drugs. The central intermediate in biosynthesis of these alkaloids are (S)-reticuline (Springob and Kutchan, 2009) and it was previously reported in many plants of Menispermaceae family (Dasgupta et al., 1979;Abbasoglu et al., 1991;Likhitwitayawuid et al., 1993;Nawai et al., 1999). (S)-reticuline is used in the preparation of drugs against cancer and malaria (Minami et al., 2008) and it also possesses cardiovascular effect (Medeiros et al., 2009). Sinomenine (cocculin), an anti-rheumatic alkaloid reported earlier from Sinomenium acutum (Zhao et al., 2012) belonging to the same family, is detected in the leaf of H. validus. Ambelline is found generally in Amaryllidaceae plants that possess antimicrobial, anti-inflammatory, anti-cancer and antioxidant properties (Nair et al., 2012). Ecgonine is a tropane alkaloid and used as a mild vasodilator and topical anesthetic. Metanephrine is a protoalkaloid with rare occurrence in nature and assumed to have sympathomimetic properties. Previously Keller et al., (1973) isolated this compound from the cactus, Coryphantha macromeris.    Choline is a quaternary ammonium compound with nutritional importance and used for liver health (Zeisel, 1992;Zeisel and da Costa, 2009;Idrus et al., 2017) and it was reported previously from Menispermaceae plants including Tinospora cordifolia.
In conclusion, the current HR LCMS analysis of the lesser known medicinally important fruit crop Haematocarpus validus revealed the presence of six alkaloid compounds in the methanolic fractions of leaf and fruit extracts.
The therapeutic potential of these alkaloid compounds support the folklore knowledge regarding this ethnomedicinal plant. Considering the enormous medicinal prospects as well as its rare and vulnerable status, this crop needs conservation, domestication and propagation in the humid and hot tropics of Asian continent for its better utilization.