Rauvolfia (PROSEA)

Plant Resources of South-East Asia
Introduction

List of species

Rauvolfia L.

Protologue: Sp. pl. 1: 208 (1753); Gen. pl. ed. 5: 98 (1754).

Family: Apocynaceae

Chromosome number: x= 11; R. serpentina: 2n= 22, 44, R. sumatrana: n= 88, R. verticillata: 2n= 22

Major species

Rauvolfia serpentina (L.) Benth. ex Kurz,
R. verticillata (Lour.) Baillon.

Vernacular names

Snakewood (En)
Indonesia: lameh
Philippines: sibakong
Vietnam: cây ba gạc.

Origin and geographic distribution

The number of species in Rauvolfia varies from 60-125, depending on literature sources. The genus is pantropical. Ten species occur in Africa and Madagascar, about 20 in tropical America, and the remainder in Asia, Australia and the Pacific. Nine species have been distinguished in the Malesian region, but as a result of new taxonomic studies this number might be reduced to 5.

Uses

Rauvolfia has been used in Ayurvedic medicine in India since ancient times to treat snake bites, mental diseases and epilepsy, and is still important. In Vietnam it is considered to be one of the most useful and effective medicinal plants. Its main use is as an anti-hypertensive agent and as tranquilizer. An extract of the root rind is considered as a highly effective remedy against high blood-pressure and to relieve the central nervous system. Besides this, it is also used to treat dysentery, diarrhoea, liver diseases, psychoses, insanity, epilepsy and snake bites and/or scorpion stings, and to stimulate uterine contraction and to promote the expulsion of the foetus. In a mixture with other plants it is sometimes used to treat cholera and fever. The leaf juice is applied locally against opacity of the cornea and to treat wounds and itch. The drug is also considered as an anthelmintic and tonic.

Rauvolfia species are used for the extraction of alkaloids, principally reserpine, but also ajmaline and ajmalicine. Reserpine is marketed for the treatment of (mild to moderate) arterial hypertension. Combination with a diuretic is classical in order to control the water and Na⁺retention that is a side effect of reserpine. The drug has been much used since the 1960s, however, because of its narrow therapeutic index (i.e. the difference between therapeutic and toxic doses is very small) and its major side effect (depression), reserpine is currently of secondary interest. More manageable compounds have become available for therapy. In psychiatric therapy, reserpine has also been replaced by synthetic products (e.g. phenothiazines and butyrofenon derivatives).

Ajmalicine is an ingredient of propietary products used to treat the psychological and behavioral problems of senility, sensory problems, cerebrovascular accidents, cranial traumas and their neurological sequelae. Another alkaloid, ajmaline is used as a remedy for heart arrhythmias in distinct cases (class 1A agent).

The lightweight wood of some Rauvolfia spp. which can reach the size of a tree is sometimes used for small objects such as knife handles.

Production and international trade

Rauvolfia roots and their preparations are used and traded in many countries and are important on the international market. Thailand appears to be the main exporter, with amounts of about 100 t/year in the second half of the 1970s, whereas Nepal exported some 30 t/year in the same period. India was a large supplier of R. serpentina roots before 1969 (on average 40 t/year), but then the export was banned by the government to help develop an extraction industry. In 1982, the price of powdered R. serpentina roots was US$ 9/kg and of reserpine US$ 0.4/g.

Properties

Commercial samples of R. serpentina drugs consist of tortuous pieces of root of up to 15 cm long and 2 cm in diameter; they are yellowish and of low density. Although at present the botanical name is Rauvolfia , the crude drug is called Rauwolfia (Rauwolfia Radix). The total alkaloid content is 0.5-3%, and over 50 different alkaloids have been demonstrated, most of them indole-type, derived from tryptophan. The alkaloids can further be classified into 4 main groups (typical analysis in parentheses): (1) yohimbane-type derivatives, e.g. reserpine (0.14%), reserpinine (rescinnamine, 0.015%), isorauhimbine (3-epirauwolscine, 0.08%), (-)-corynanthine (rauhimbine, 0.03%), deserpidine, yohimbine and corynantheine; (2) heteroyohimbane derivatives, e.g. serpentinine (serpentidine, 0.13%), serpentine (0.08%), raubasine (ajmalicine, 0.02%), reserpiline and alstonine; (3) sarpagane derivatives, e.g. sarpagine (raupine, 0.02%); and (4) dihydro-indole (ajmalane) derivatives, e.g. ajmaline (0.1%). Standardized Rauwolfia powder has a total content of 0.15-0.20% alkaloids of the reserpin-rescinnamine group, calculated as reserpine, and analysed according to the USP XXII. Furthermore, several spectrophotometric and extraction-photometric methods have been developed for determining Rauvolfia alkaloids, e.g. reserpine, serpentine and ajmaline. These methods are comparatively simple and accurate.

Of the Rauvolfia alkaloids, 5 are used in medicine: reserpine, rescinnamine, deserpidine, raubasine (ajmalicine) and ajmaline. There are several patented methods available for the extraction of the main component reserpine.

Reserpine is a sympatholytic agent acting indirectly on the peripheral (noradrenergic) and central (noradrenergic and serotonergic) nerve terminals. By inhibiting the Mg²⁺and ATP dependent transport of e.g. noradrenaline (norepinephrine) it amplifies the breakdown of this substance by monoamino oxidases and catecholamine transferases. The resulting depletion of catecholamines on the peripheral level induces a lasting drop in blood pressure and heart rate. Sedative and neuroleptic activity is caused by central neurotransmitter depletion. Contra-indications for using reserpine are depression, combination with monoamino-oxidase inhibitors or laevodopa, peptic ulcer, and hypersensitivity to the alkaloid. Side effects of the medication include drowsiness, nasal congestion, salivary and gastric hypersecretion, paradoxical anxiety, depression and retention of water and Na⁺(the latter may be overcome by co-administration of a diuretic). Overdose may cause respiratory depression, bradycardia, hypotension, confusion, tremors, myosis, convulsions and gastro-intestinal distress. Oral administration of R. serpentina extract has caused blood and urine glucose to decrease in cats and diabetic patients. Reserpine has been shown to enhance the hypoglycaemic effect of insulin and the hyperglycaemic effect of adrenalin, and has inhibited the physiological hyperglycaemic response in diabetic patients. Reserpine did not show genotoxicity and was unable to induce reverse mutation and recombinational mitotic events (crossing-over and gene conversion) in yeast strains.

Rescinnamine (reserpinine) is a reserpine analogue containing a trimethoxycinnamic rather than a trimethoxybenzoic-acid residue. Deserpidine (11-demethoxyreserpine) lacks a methoxy function at position 11 of the reserpine molecule. Both alkaloids have the same effects as reserpine, and can be used to treat the same conditions, although their side effects are reported to be less pronounced.

Raubasine (ajmalicine) is an α-adrenergic blocking spasmolytic which, at high doses, reverses the effects of adrenalin (epinephrine), and moderates the activity of the vasomotor centres, especially in the brain stem. It causes a transient increase of the blood flow to the brain, and is slightly anxiolytic.

Ajmaline is an anti-arrhythmic (class 1A group), which substantially decreases the rate of depolarization of atrial and ventricular cells. Its toxicity has limited its uses to some specialized cases.

The alkaloids reserpine, spegatrine and verticillatine have been reported in R. verticillata. Spegatrine has been shown to be an α-adrenergic blocker, whereas verticillatine exhibited ganglionic blocking activity. Clinical application of verticillatine showed significant therapeutic effect in treating severe cases of hypertension, with few side effects.

R. serpentina powder at a concentration of 0.25% has been found to be very effective in protecting grain against Rhizopertha dominica. The roots also exhibit plant growth inhibition and uterine stimulant, antihyperglycaemic, and dopamine receptor blocking activity. The leaf extract showed fungitoxicity; it reduced growth of Sclerotium rolfsii significantly. An extract of R. serpentina markedly decreased the number of local lesions and systemic infection caused by brinjal necrotic mosaic virus on aubergines.

Adulterations and substitutes

Apocynaceae other than Rauvolfia, for example Catharanthus roseus (L.) G. Don, have similar or related alkaloids with similar applications.

Description

Shrubs or small to medium-sized trees up to 30 m tall, often candelabra-shaped, with latex in branchlets but not in bark; bark smooth, rough, fissured or scaly.
Leaves verticillate in whorls of 3-4(-5), rarely opposite, simple and entire, short-petioled with axillary glands on the petiole, exstipulate.
Inflorescence a terminal, peduncled cyme, sometimes seemingly axillary.
Flowers actinomorphic, bisexual, small, 5-merous; calyx deeply divided, with overlapping lobes; corolla salver-shaped, with cylindrical or campanulate tube, sometimes ventricose in or above the middle, with long hairs inside in the upper half, throat usually constricted, often hairy, lobes twisted to the left in the bud, white or greenish-white to pink, tube often reddish outside; stamens inserted in the widening of the corolla tube, alternating with corolla lobes, filaments very short, anthers not or hardly exserted, free from stigma, medifixed, acute; disk annular or cupular; ovary, superior, with 2 carpels which are free or connate, style 1, glabrous, stigma provided at the base with a collar and with a bifid apical cusp.
Fruit consisting either of 2 free drupelets or a single, entire or bilobed drupe with 1-2 tuberculate pyrenes, usually about 1 cm in diameter, ripening blackish.
Seeds laterally compressed, obliquely ovate or elliptical, with a large embryo; endosperm fairly abundant.
Seedling with epigeal germination; cotyledons leafy, green; first 2-3 nodes with decussate leaves, subsequent leaves whorled.

Growth and development

Ramification in Rauvolfia is determined by the verticillate leaves; branches terminate in 2-5 elements, consisting of branchlets or inflorescences. The number of elements is the same as the number of leaves in the whorl. This results in an umbellate ramification and a candelabra-shaped habit.

R. serpentina and R. verticillata flower throughout the year in Peninsular Malaysia. The flowers are pollinated by insects like small bees and flies.

Other botanical information

Full-grown flowers and ripe fruits are indispensable for a reliable identification of Rauvolfia. R. javanica, R. reflexa and R. sumatrana are difficult to distinguish, and might be considered as one polymorphous species.

Rauvolfia resembles Ervatamia and Kopsia, but Ervatamia differs in its opposite leaves and seeds with red or orange sarcotesta, and Kopsia in its opposite leaves, corolla segments overlapping to the right and disk consisting of 2 scales alternating with the carpels.

The roots or root rind of R. cambodiana Pierre ex Pitard, R. chaudocensis Pierre ex Pitard, R. indochinensis Pichon and R. vietnamensis Ly are used in Vietnam to treat high blood pressure, and sometimes also to treat dysentery and as antibiotic and antiseptic. R. vomitoria Afzel. is an important species from West and Central Africa with high alkaloid concentration. R. tetraphylla L. is from tropical America. Both species are used for industrial alkaloid extraction; R. tetraphylla is cultivated very locally in gardens in India, Vietnam and China, R. vomitoria in Vietnam.

Ecology

Rauvolfia species occur scattered in forest, often in secondary forest, and scrub vegetation. Several tolerate shade well, but some (e.g. R. serpentina ) occur especially in more open places, e.g. in forest edges and along rivers. Slightly acid soils (pH 5-6.5) are favourable, but some Rauvolfia spp. also grow well on limestone soil. Experiments with R. serpentina grown in different soil media in the Philippines showed no significant difference in root production.

Propagation and planting

R. serpentina is usually propagated by seed, although stem and root cuttings can also be used. The germination rate can be very low, therefore it is recommended to select mature and heavy seeds and to sow them within 6 months after ripening. In Vietnam, fruits are soaked in water for 12 hours, they are then crushed and the seeds are cleaned and subsequently soaked in warm water (40-45°C) for a further 12 hours. The seeds are sown in nursery beds, and start germinating within 3 weeks. Seedlings 10-12 cm tall are usually transplanted during the rainy season at a planting distance of 45 cm × 30 cm. A planting distance of 50 cm × 50 cm is recommended for R. verticillata. Propagation of R. serpentina in India gave a success rate of 40-65% from stem cuttings, 50-80% from root cuttings; a success rate of up to 90% has been obtained in Vietnam under careful management.

Successful in vitro propagation techniques have been developed for R. serpentina. Sterilized shoot tips can be cultured in Murashige and Skoog medium containing 3% sucrose. Best shoot response was obtained in the presence of 0.5 mg/l naphthalene acetic acid and 2 mg/l benzyladenine, with 15-20 shoots arising from one shoot tip. For rooting it is essential to replace benzyladenine by kinetin. When the resulting plantlets were planted in the field the survival rate was 60% and the plantlets were cytologically stable. Plantlets have also been successfully regenerated from shoot cultures of R. serpentina initiated from auxiliary meristems on medium containing benzyladenine (4.5 μM) and naphthalene acetic acid (0.5 μM). Rooting was initiated in White's basal medium supplemented with 0.5 μM naphthalene acetic acid. The resulting plants were similar to normal ones in their morphological characteristics and chemical constitution, but produced more biomass.

Nodal cultures of R. serpentina could be maintained for 9 months at 25°C on a standard Murashige and Skoog medium. Low-temperature incubation of in vitro cultures appeared highly promising because cultures exhibited normal health even after 15 months of storage at 15°C. Temperatures of 5-10°C were found to be deleterious to the growth of the cultures.

The total indole alkaloid content of R. serpentina roots from plants regenerated from stem and root callus was slightly higher than in the parental stock, but the content of ajmaline, serpentine and reserpine was lower.

In vitro production of active compounds

Cell suspension cultures of R. serpentina have proved to be an excellent source of the enzymes involved in the biosynthesis of the alkaloids of the ajmalane and sarpagane class. Moreover, cell suspension culture is one of the most efficient methods for indole alkaloid formation. So far, about 30 different indole alkaloids have been isolated and identified from cultivated Rauvolfia cells. Alkaloid production under optimum conditions ranges from micrograms to grams per litre medium (e.g. yields of up to 1.6 g/l of raucaffricine have been obtained). A cell suspension culture of R. serpentina continuously treated with hydroquinone produced up to 18 g/l of p-hydroxyphenyl-O-β-D-glucoside (arbutin), which is the highest transformation rate ever observed with a plant cell culture system for a single natural product.

Raucaffricine (vomilenine-galactoside) has been shown to be the major indole alkaloid of cell suspension cultures of R. serpentina grown in alkaloid production medium. This compound is converted by an enzyme to its aglycon vomilenine, which has a key function in the biosynthesis of ajmaline. A higher content of intracellular indole alkaloids of the ajmaline type was found in a transgenic hairy root culture in liquid Murashige and Skoog medium than in the leaves and roots of the intact plant. It has also been found that phytosterols, including stigmasterol, β-sitosterol and cholesterol, are produced in callus tissue cultures.

It has been demonstrated that there are significant genome rearrangements in R. serpentina callus cultivated for a long time. These can occur at early stages of cultivation, but the process of subsequent subculturing in vitro leads to more significant genome changes. A highly productive cell line was found to be a partially synchronized myxoploid stable cell population in which tetraploid and multiploid cells and cells with a low level of structural mutations of the chromosomes predominated. The content of indole alkaloids in the tissue was positively correlated with the increase in the frequency of tetraploid and multiploid cells.

Hybrid cell suspension cultures have also been generated (e.g. from R. serpentina and Rhazya stricta Decne., and from R. serpentina and Vinca minor L.). Alkaloids not previously detected in the parental cell cultures may be formed in the hybrid culture, and in a cell line of a R. serpentina and Vinca minor hybrid a 10-fold increase in raucaffricine accumulation was observed relative to the parental Rauvolfia strain.

Husbandry

Regular weeding and hoeing (2-3 times during the growing season in India) is needed to maintain satisfactory development of the roots. A top dressing of ammonium sulphate given after weeding will promote the development of a vigorous stand. R. verticillata grows more vigourously than R. serpentina and will soon shade out weeds. In Malaysia, R. serpentina has been found very sensitive to magnesium deficiency, causing severe chlorosis; application of moderate amounts of potash fertilizers is therefore recommended. In India, application of farmyard manure and compost showed a beneficial effect on the growth of R. serpentina.

Diseases and pests

In India, wilting caused by Fusarium is the most serious disease in cultivated R. serpentina, followed by Alternaria leaf blight, powdery mildew, mosaic virus disease and root-knot disease, resulting in stunted growth. Leaf spot and blight caused by Rhizoctonia solani result in premature defoliation in the rainy season; weeding up to a distance of 45 cm from the plants effectively controlled the pathogen, as weeds serve as host. Young branches are sometimes infected with green bugs.

Harvesting

The roots of R. serpentina are usually harvested from the wild, although it is cultivated to a limited extent, e.g. in India. When cropped, the roots are harvested after 1.5-3 years. Care should be taken to keep the root bark intact as it has a high alkaloid content. In Vietnam, it is recommended not to gather the roots of R. verticillata in spring because the concentration of active compounds is low then. Preferably, only roots with a diameter over 3 cm should be collected, leaving small-diameter roots for a next harvest.

Yield

The average yield in R. serpentina plantations in India is 2 t/ha when roots are harvested 15 months after planting, and 36 t/ha when harvested after 3 years. However, harvesting after 15 months was found to be most lucrative. Under favourable soil conditions in Vietnam, 3-year-old plants produced on average 50 g of dried root rind.

In a small-scale experiment in Peninsular Malaysia, the average yield of dried root per R. verticillata plant was 200 g 1.5 years after planting, 600 g after 2 years and 930 g after 3 years.

Handling after harvest

Roots of Rauvolfia are usually cut into pieces 10-15 cm long. To prevent fungus infection they should be dried before storage to approximately 8% moisture content. Dried roots are usually packed in jute bags or bales, smaller quantities for regional markets are packed in polyethylene bags of 2-5 kg. Material to be packed in polyethylene bags must be adequately dried.

Genetic resources and breeding

Natural populations of R. serpentina have been overexploited in many regions, particularly because roots are usually harvested, which destroys the plants. The species may now be extinct in Sri Lanka, due to intense exploitation, and in India and Java it has become rare. Several Rauvolfia species are threatened with extinction in Vietnam, e.g. R. chaudocensis, R. indochinensis and R. vietnamensis. These valuable plant resources must be protected by ex situ and in situ conservation. There are no known germplasm collections.

Prospects

The use of Rauvolfia in galenic preparations and the use of reserpine have declined significantly in recent decades in industrialized countries because of their strong side effects and the availability of more effective alternatives. Moreover, reserpine has been suspected of having carcinogenic effects (causing breast neoplasm) and, although this effect has not been confirmed, its use has declined considerably and is unlikely to regain its former level. In developing countries, products based on Rauvolfia are still in demand owing to their easy availability and comparatively low prices, but it is expected that in the South-East Asian countries they will be gradually replaced by modern alternatives. As long ago as 1958, commercial cultivation of Rauvolfia was considered uneconomic in Malaysia, mainly because of the apparently adequate stocks of Indian origin, although trials had shown that it could be brought into cultivation at short notice. However, continued research might reveal new possibilities.

Literature

Allen, E.F., 1958. Notes on the cultivation of Rauwolfia in Malaya. Malayan Agricultural Journal 41(2): 100-105.
Banerjee, N. & Sharma, A.K., 1983. Cytotaxonomy, tissue culture and alkaloids of Rauwolfia L. Nucleus 26(3): 197-207.
International Trade Centre UNCTAD/GATT, 1982. Markets for selected medicinal plants and their derivatives. Geneva, Switzerland. pp. 106-112.
Markgraf, F., 1984. Florae Malesianae Praecursores LXIV. Apocynaceae VI. Rauvolfia. Blumea 30: 157-167.
Mukhopadhyay, S., Mukhopadhyay, M.J. & Sharma, A.K., 1991. In vitro multiplication and regeneration of cytologically stable plants of Rauwolfia serpentina Benth. through shoot tip culture. Nucleus 34(3): 170-173.
Roja, G. & Heble, M.R., 1996. Indole alkaloids in clonal propagules of Rauwolfia serpentina Benth. ex Kurz. Plant Cell, Tissue and Organ Culture 44(2): 111-115.
Ruyter, C.M., Akram, M., Illahi, I. & Stöckigt, J., 1991. Investigation of the alkaloid content of Rauwolfia serpentina roots from regenerated plants. Planta Medica 57(4): 328-330.
Sarin, Y.K., 1982. Cultivation and utilization of Rauvolfia serpentina in India. In: Atal, C.K. & Kapur, B.M. (Editors): Cultivation and utilization of medicinal plants. Regional Research Laboratory, Council of Scientific and Industrial Research, Jammu-Tawi, India. pp. 288-294.
Schütte, H.-R., 1991. Secondary plant substances: monoterpene indole alkaloids. Progress in Botany 52: 84-96.
Sharma, N. & Chandel, K.P.S., 1992. Low-temperature storage of Rauvolfia serpentina Benth. ex Kurz: An endangered, endemic medicinal plant. Plant Cell Reports 11(4): 200-203.

Selection of species

Authors

Tran Dinh Ly & Pham Duy Mai