Protective effects of Indian medical plants against cyclophosphamide neutropenia.
The most common cause of death in patients with malignancies is infection. This is due to a combination of factors, prime among them being the immunosuppression induced by the cancer chemotherapeutic drugs. A recent development in cancer chemotherapy has been the concurrent use of agents like lithium with immunomodulating effects to prevent adverse effects of cancer chemotherapeutic agents on bone marrow. Several substances like glucans, ubiquinone, muramyl dipeptide and its derivatives, endotoxin and bacterial products like C. parvuan or BCG have been reported to enhance host defence. However, a safe and effective drug is yet to be found.
Indigenous plant materials offer a large potential source from which active and safe immunomodulators could be obtained. Asparagus racemosus (AR), Tinospora cardifolia (TC) and Withania somnifera (WS) are plants used in Ayurveda, and Indian Traditional System of Medicines as general tonics or Rasayanas. We have found in our earlier studies that pretreatment with these agents protected rats against mixed bacterial abdominal sepsis. This was accompanied by a significant increase in the phagocytic activity of peritoneal macrophages. As activated macrophages are known to secrete mediators which cause leucocytosis, the present study was carried out to identify whether these 3 plants protected mice against cyclophosphamide (commonly used anticancer drugs) induced neutropenia.
The study was conducted in 50 adult Swiss albino mice of either sex, divided into 5 groups depending on the drug and dose used, as shown in [Table 1]. Ail animals were pretreated for 15 days with the drug, or vehicle in case of the control group. The roots of AR, stems of TC and whole plant of WS were used in decoction form. All drugs were given orally by intragastric tube. The protocol of pretreatment was followed as prescribed in Ayurveda. Ayurveda emphasises maintenance and promotion of health more than curative aspects. Hence a protocol of pretreatment is recommended.
Cyclophosphamide was injected in a single dose of 200 mg/kg subcutaneously after 15 days of treatment. This day was labelled as day zero.
Blood was collected by retro-orbital venous sinus puncture with a capillary tube and processed as follows. Total and differential white blood cell counts were performed prior to (on day 0) and on days 3 and 7 after injection of cyclophosphamide.
In one group of animals, we administered Asparagus racemosus (100 mg/kg daily) from the day of cyclophosphamide injection. This group was included to verify whether pretreatment was a necessity or not.
The total WBC and absolute neutrophil counts, in treated group were compared with the values of the control group. The unpaired 't' test was used to calculate statistical significance. Only p < 0.0125 was considered as statistically significant, applying the Bonferroni method to take into account the multiplicity factor which is introduced while applying the 't' test to compare more than 2 groups.
Injection of a single dose of 200 mg/kg cyclophosphamide subcutaneously produced a fall of 43% in total leucocyte count and 25.18% in absolute neutrophil count on the third day. A rebound increase in total WBC count and neutrophil count occurred on the 7th day.
The animals treated for 15 days with (100 mg/kg/day) AR, TC and WS showed a significant leucocytosis with predominant neutrophilia. The leucopenia and neutropenia which occurred in control animals following cyclophosphamide was less marked in the treated animals [Table 2].
A point to note is that the WRC counts in all treated animals never went below the normal range (6000-8000 cells/mm) after injection of cyclophosphamide. A rebound increase in the counts occurred on the 7th day of these animals too. No mortality was found in the groups given Withania somnifera, while 1 animal out of 10 died after cyclophosphamide injection in the other groups.
In the last group where AR was co-administered with cyclophosphamide and treatment continued after cyclophosphamide, no leucocytosis or protection against neutropenia occurred.
We have demonstrated that the 3 Indian medicinal plants Asparagus racemosus, Withania somnifera and Tinospora cordifolia protected mice against cyclophosphamide induced neutropenia.
Pretreatment for 15 days with these drugs produced a striking leucocytosis with a predominant neutrophilia. The leucopenia and specifically neutropenia induced by cyclophosphamide was significantly reduced by both Withania somnifera and Tinospora cordifolia. The total WBC and absolute neutrophil counts following Asparagus racemosus had risen very high (18800 ± 2001, 13366 ± 501.96 respectively) so that the % fall after cyclophosphamide (63.77% total counts, 58.13% neutrophil counts) was greater than that in the control group (43.78%, 25.18% respectively). However, the counts never went below the normal range.
It is further evident that pretreatment is a necessity.
The results in our study open up exciting possibilities for the use of plant products in the prevention of adverse bone marrow effects associated with cyclophosphamide. Acute and subacute toxicity studies that we have conducted (unpublished) indicated that no significant toxicity occurred with this dosage regimen. There was marginal and reversible hepatosplenomegaly without significant histopathological changes. These drugs Asparagus racemosus (Shatavari), Tinospora cordifolia (Guduchi) and Withania somnifera (Ashwagandha) are plants already widely used, even in humans by Ayurvedic physicians.
These plants produce the neutrophilia and leucocytosis probably by activating macrophages. Activated macrophages secrete a large number of substances including a colony stimulating factor and interleukin 1. These substances are known to cause leucocytosis., We have found that the colony stimulating activity of serum from mice receiving these drugs is increased (unpublished). It appears therefore that the activation of macrophages produces the protection of the mice against cyclophosphamide neutropenia.
These results have opened up new vistas in the area of immunopharmacology of Indian medicinal plants.
Extrapolation of animal data to a clinical setting is not always appropriate. Yet, the exciting results we have obtained indicate that human studies are needed to evaluate these plants for their immunomodulating properties.
This work was supported by a grant in aid from the K.E.M. Hospital and Seth G.S. Medical College, Research Society, Parel, Bombay 400 012.