The British Homoeopathic Journal July 1988. Vol. 77. pp. 147-151

Pancreatic beta-cell regeneration a novel anti-diabetic action o f Cephalendra indica mother tincture

D. P. RASTOGI*, A. C. SAXENAt , SUNIL KUMAR$

Abstract Cephalendra indica 0 (41% v/v alcoholic extract of the wild variety of Cephalendra indica Naud.), on regular administration in doses ranging from 25 ~tml to 75 Ixml/100 g of body weight (gbw) by the oral or intraperitoneal (ip) route produced a significant fall in blood sugar level in alloxan-induced diabetic rats. Biochemical studies showed stabil- ization of blood sugar level in 70% of cases at fourteen to twenty days after withdrawal of the drug. Histopathological studies revealed regeneration of pancreatic [3 cells. The hypothesis is that the drug acts through the hypothalamo-hypophysial-pancreatic axis, producing selective regeneration of 13 ceils. The drug may indirectly release inhibitory factors from hypothalamic neurons, inhibiting the secretion of growth hormone and triggering insulin secretion from [3 cells. The therapeutic action of the drug on pancreatic [3 cells and lack of acute and subacute toxicity may open up new prospects in the treatment of diabetes mellitus.

Introduction Treatment of diabetes has been reviewed by many researchers. 1-4 The causative factor of diabetes has attracted considerable investigation over the centuries and still remains uncertain. Polyuria and wasting are the dominant clinical features: ' A melting down of the flesh and limbs into urine' (Aretaeus, 2nd century AD) , and it was long thought to be due to kidney disease. Recently, extensive data have accumulated to establish that renal disease is secondary to glycosuria. ~6 Agrawal and Bapat 7 on the basis of their piqure experiments believe that diabetes is due to disease of the central nervous system. There are undoubtedly many different causes of diabetes. Advances in genetics, the concept of neuroendocrinology, understanding of major histocompatibility complex and developments in immunology have considerably advanced our understanding of the nature of the disease.

We have experimented with mother tinc- ture of Cephalendra indica in diabetes mellitus experimentally produced by alloxan

*D. P. Rastogi, Director, Central Council for Research in Hom0eoapthy (Min. of Health & Family Welfare, Govt. of India). ?A. C. Saxena, Project Director, Homcepathic Drug Research Institute, Sector B-1433, Indira Nagar, LUCKNOW-226 016. :~Sunil Kumar, Research Officer (P), HDRI.

(C4H2N204H20,M.W. 160.09, purity 98.5 %). In the present investigation, an attempt has been made to locate the mode of action of the drug with particular emphasis on [3 cell regeneration and its neuroendocrinological control.

Materials and methods One hundred and eighty albino rats of either sex weighing 240+12 g were selected after ac- climatizing to standard laboratory conditions for fifteen days. Water was allowed ad-libitum. Photo-period L/D (10 light hours/14 dark hours) was also maintained. The acclimatized animals were subjected to quantitative analysis of blood sugar by the Folin and Wu method by taking a 0.5 ml blood sample from the tail vein or through cardiac puncture and measuring absorbance at 620 nm wave length in a Beckman Model 35 spectrophotometer.

Diabetes mellitus was induced by intra- peritoneal injections. Three doses of 10 to 12 rag/100 gbw, at intervals of seven days, of alloxan dissolved in distilled water, were admin- istered after twelve hours fasting. Blood sugar estimations were done to confirm the establish- ment of diabetes mellitus. The diabetized ani- mals were divided into six groups for in-vivo and in-vitro studiesl The short-term experiment was conducted over twenty one days and the long-

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148 British Homoeopathic Journal

Figure 1: Degenerative changes with prominent beta cell necrosis after alloxan treatment. Gomori's aldehyde fuchsin

x 500.

Figure 2: Cephalendro indica 0 treated after 30th day (25 uml/100 gbw), with prominent 13 cell restoration. Gomori's

aldehyde fuchsin •

Figure 3: Cephalendra indica r treated after 30th day (75 uml/100 gbw) with large number of [3 cells in islet of

Langerhans. Ehrlich's haematoxylin and Eosin •

Figure 4: Cephalendra indica ~ treated after 30th day (25 ~tml/100 gbw) S.S. of brain, passing through hypothala- mus. Non-secretory activity with hypotrophic neurons and

significant GH-IF. Gomori's aldehyde fuchsin x500.

Figure 5: 41% v/v alcohol fed control after 30th day. S.S. of brain, passing through hypothalamus. Perceptibile secretory activity with prominent hypertrophied neurons and indicating

GH-RF. Gomori's aldehyde fuchsin x500.

Volume 77, Number3, July 1988 149

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term experiment over forty five days. The paral lel studies were conducted with tolbutamide (Rastinon), glibenclamide (Daonil) and insulin. A market sample of Cephalendra indica ~ was studied in addition to a tincture prepared in the Homceopathic Pharmacopoeia Laboratory, Ghaziabad, and the therapeutic efficacy of the drug compared with the 41% v/v alcohol control, since the ~ is prepared in 41% v/v alcohol as specified in the Homceopathic Pharmacopoeia of India. Both the oral and the i,p. route of drug administration were attempted. The normal control group was given 0.9% w/v physiological saline. The diabetized rats were divided into groups of ten as follows:

The long4erm experiment was conducted for forty-five days, but the drug was only admin- istered for the first thirty days. After that, drug administration was stopped and the animals assessed for blood sugar stabilization.

The blood sugar estimation was done at twelve hours fasting and two hours postprandial on the first, fifth, seventh, fourteenth, twenty first and thirtieth days. The histopathological studies were conducted on a 25% sample of the experimental animals. The brain, pituitary gland, pancreas, liver, kidney etc., were isolated and processed for histopathological studies. These organs/tissues were isolated by decapita tion of animals, after which the entire retro- peritoneal fat containing pancreatic tissue was dissected out and fixed in freshly prepared Bouin's fluid. The tissues were cut into two to four #m thick sections and stained in haematox- ylin eosin and Gomori aldehyde fuchsin stain with aqueous light green 0.6 g + chromotroph- 2 R 0.5 g + orange G 1.0 g + glacial acetic acid 1 ml/100ml of water as counter stain. The [3 cells per islet area in cross section (mm x 350) were counted under a light microscope.

II

Results ~ Experimental studies revealed that regular u administration of Cephalendra indica 0 for

.~ thirty days in micro doses ranging from 25 gml. to 75 ,aml./100 gbw produced a slow and steady fall in blood sugar level, and hypoglyc~emic activity came into prominence after the tenth to fifteenth day of treatment. The normal control and alcohol fed control groups continued to be hyperglyca~mic (Table 1.)

o The beta cell counts and blood sugar levels in various groups of alloxanized rats are given in

It Table 2. It is evident from the table that alloxan - brings about necrosis of ~3 cells and markedly

150

TABLE 2

British Homceopathic Journal

Influence of Cephalendra indica ~ on pancreatic beta cells and blood sugar levels in alloxanized rats (Mean_+S.E.)

Blood sugar Normal Control level/f3 cell control 41% v/v counts 0.9% w/v alcohol

Cephalendra indica r administration on 30th day in per 100 gbw

25 gmI 50 gml 75 gml 0.1 gml P

Blood sugar 2 5 5 - + 3 . 2 266_+20 80+2.0 100_+1.8 90-+2.7 115+3.2 A I : B 1 NS* level (fasting A~:C~ <0.001 in mg/dil.) A t B~ C~ D~ E~ Fj A~:Dt <0.001

A]:E] <0.001 AI:FI <0.001

Beta cell 20• 18_+3.2 36• 30+2.8 32+5.0 26• A2:B2 NS* counts per islet A2:C 2 <0.001 area in cross A2:D2 <0.001 section Ai:E 2 <0.001 ( m m 2 X 350) A2 B2 C2 D~ E 2 F, A 2 : F 2 <0.001

*NS = Not significant. All statistical values calculated by Student t test.

TABLE 3 Hypoglycaemic activities of Cephalendra indica r Stabilization of blood sugar level after withdrawal of drug

Drug/vehicles Route of Days at which blood Effective doses Duration of stabilization Percentage of admn. sugar level comes to per 100 gbw of blood sugar level stabilization after

normal withdrawal of drug ED 50 & ED 100 mean value analysis

41% v/v alcohol oral/i.p. Exhibited None effective Never attained normal Nil control hyperglycaemic action level

Cephalendra oraI/i.p. Attained normal level Effective does are 14 to 20 days (in 30 days 70% indica ~ usually after 15 days of 25 gml to 75 gml, regular admn. of drug)

regular drug admn. at a with 25 gml most No toxicity (30 days 70% dose level of 25 gml to effective regular frug admn.) 75 gml/100 gbw depending on blood sugar level

'\

reduces the n u m b e r along with an increase in blood sugar level (Fig. 1). The Cephalendra indica ~ t rea ted animals exhibi ted b lood sugar level and 13 cell counts within normal range at a dose level ranging f rom 25 gml to 75 Bxml/100 gbw (Figs. 2 & 3).

Histological examina t ion of the brain revealed cer tain hypotha lamic neu rons having specific s t ructural peculiari t ies and conta in ing secretory mater ia l and p rominen t ly nuc lea ted with bipolar axons. These s t ructures are identi- fied by special techniques of t inctorial affinities (developed in our Labora tory) . These neurons are juxtasomal with b lood vessels and respon- sible for the secret ion of releasing factors (RF) or inhibi tory factors (IF) for growth h o r m o n e s (GH) . Excessive secret ion of G H probab ly inhibits insulin secret ion and produces d iabetes melli tus by damaging the [3 cells. However , in the present exper imen t it was no ted that the drug had b rough t abou t hypo t rophy in hypoth-

alamic neurons (Fig. 4) and p robab ly re leased the IF which in tu rn inhib i ted growth h o r m o n e secret ion f rom the adenohypophys is . This may have resul ted in the res tora t ion of 13 cells counts ' and regenera t ion , whereas the control groups had shown hype r t rophy and degranu la t ion in hypo tha lamic neurons (Fig. 5).

Table 3 shows the f requency of b lood sugar level s tabi l izat ion af ter wi thdrawal of the drug. It was observab le tha t the b lood sugar level was stabil ized for four teen to twenty days in 70% of cases in the Cephalendra indica ~ t rea ted group, when compared to control animals.

The parallel studies with to lbu tamide , glib- enc lamide and insulin conducted unde r identical l abora tory condi t ions showed a sudden fall in b lood sugar level, which however rose again on wi thdrawal of the drug, (Table 1).

Conclusions T h o u g h mitosis in 13 cells is possible, 8 no docu-

Volume 77, Number3, July 1988 151

mented report of drug-induced 13 cell regeneration seems to be available. This is the first study of its kind with a homeeopathic drug. Cephalendra indica O. However, the present observations are in close conformity with the findings of Chakravarthy et al. with Pterocarpus marsupium Roxb., 9,1~ which causes selective regeneration of 13 cells of alloxan-damaged pan- creas and protects the 13 cells against the necrotic effect. There is a close parallelism between the slow and steady fall in blood sugar level and perceptible increase in 13 cell counts and graded inactivation of the hypothalamic neurons (hypo- trophy) in the Cephalendra indica 0 treated group, thus, the in-vitro studies suggest a prob- able neuroendocrinological mechanism of Cephalendra indica 0 in stabilizing the blood sugar level.

The experimental and clinical potential of this novel antidiabetic drug is specially significant because it did not show acute or sub-acute tox- icity even in fairly large doses, i.e. 0.1 ml to 0.5 ml/100 gbw. Blood pressure screening has shown hypotensive response at a dose level 0.1 ml/100 gbw. However, we have evaluated the stability of blood sugar levels for eighteen days after withdrawal of the drug. Further investiga- tions in this area will be rewarding in order to confirm neuroendocrinological involvement through hormonal assay of corresponding hypo- physial component.

Acknowledgements The authors offer their grateful thanks to Dr P. N. Verma, Director, Homoeopathic Pharmacopoeia Laboratory and Pro-

ject Officer, Drug Standardisation Unit, Ghaziabad, for extending laboratory facilities and technical support.

References 1 Anderson JW, Herman RH. Treatment of reactive hypo-

glyc~emia with sulfonylureas Am J Med Sci 1971; 261: 16-23.

2 Clayman CB. Tolbutamide revisited. JAm MedAss 1974; 228: 1523.

3 Widstrom A, Cerasi E. On the action of tolbutamide on man. I. Role of adrenergic mechanisms in tolbutamide induced insulin release during normoglyc~emia and induced hypoglyc~emia. Acta Endocrinol (Copenh). 1973; 72: 506-518.

4 Williams RH, Porte D. The pancreas. In Textbook of Endocrinology 5th ed, pp. 502-626. (Williams RH ed.) Philadelphia: WB Saunders Co. 1974.

5 Hartroft WS, Wrenshall GA. Correlation of beta cell gran- ulation with extratable insulin of the pancreas. Studies in adult human diabetics and nondiabetics. Diabetes 1955; 4: 1-7.

6 Van Wyck JJ, Underwood LE, Hintz RL, Voina S J, Weaver RP. Chemical properties and some biological effects of human somatomedin. In Advances in Human Growth Hormone Research (Raiti s. ed.) Department of Health, Education, and Welfare Publication No. (NIH) 74-612. US Government Printing Office, Washington, DC, 1973: 25-45.

7 Agarwal GC, Bapat SK. Indian J Med Res 1977; 66: 323- 330.

8 Ham AW, System of Endocrine Glands Histology. 7th edn, p.837. Philadelphia and Toronto: J. B. Lippincon Com- pany 1974.

9 Chakravarthy BK, Gupta S, Gambhir SS, Gode KD, Pan- acreatic beta cell regeneration--a novel antidiabetic mech- nism of Pterocarpus marsupium, roxb. lnd J Pharrn 1980; 12: 123-127.

10 Chakravarthy BK, Gupta S, Gambhir, SS, Gode KD, The prophylactic action of (-)epicatechin against alloxan induced diabetes in rats. Life Sci 1981; 29: 2043-2047.