Fatemeh Zal MS*, Mohammad Vasei MD**, Mojgan Rasti MS*, Mahmood Vessal PhD*^•

The blossoms of a related species, Teucrium chamaedrys or germander, have also been used in France for the preparation of herbal teas and capsules and were employed in folk medicine for the treatment of obesity.³ Several cases of hepatotoxicity have been reported as a result of T. chamaedrys consumption in the form of tea or capsules.^3,4 Loeper, et al⁴ showed that an intragastric administration of the lyophilisate (1.25 g/kg) of T. chamaedrys to mice would produce midzonal liver necrosis after 24 h. They concluded that a cytochrome P₄₅₀3A metabolite of one of the constituents of this plant (furano neoclerodane diterpenoids) probably acts as the major hepatotoxic factor. The aqueous extract of the dried aerial parts of T. polium in bloom is being used in many Iranian provinces, particularly in Kerman, as an antidiabetic agent. In 1995, Mattei, et al⁵ reported a case of severe liver failure caused by a herbal medicine containing T. polium, for whom liver transplantation was performed. Several neoclerodane diterpenoids, the probable hepato-toxic precursors of this herb have been reported to be present in the aerial parts of the plant.^6-15 This study, which reports the histopathological changes in the liver induced by T. polium in streptozocin-induced diabetic rats, is the first report of a series of investigations performed to evaluate the antidiabetic potential of this herbal medicine.

Reagents used in this study included streptozocin (Upjohn Co., Kalamazoo, MI, USA) and the enzymatic kit for glucose determination (Pars Azemoon Co.,Tehran, Iran). All other reagents were of analytical grade and obtained through other commercial sources.

The dried aerial parts of T. polium were purchased from herbalists in Kerman and were authenticated by the Center for Research on Natural Resources and Livestock (Ministry of Agricultural Jehad, Isfahan, Iran) as Teucrium polium; L.

The cleaned aerial parts (90 g) were suspended in one liter of water for an hour prior to simmering on a boiling water bath for another hour. The suspension was cleared upon passing through several layers of cheese cloth and freeze-dried (Heto Dry Winner, Model DW3). Approximately 20 g of dried powder was obtained from every 90 g of the original plant. The dried powder was dissolved in distilled water at a concentration of 1 g/mL. This preparation which is referred to as the aqueous extract and corresponds to 4.5 g of the original aerial parts per mL is employed as the hypoglycemic agent throughout this study (see below).

Four groups of adult male Sprague-Dawley derived rats (5 rats per group) with a weight ranging from 200 to 220 g were used as experimental animals. Each group was separately housed in a cage and fed a rat chow diet (Pars Dam Co, Tehran, Iran) and water ad lib throughout this study. Two groups of the rats were injected with streptozocin (40 mg/kg body weight) two weeks prior to the initiation of treatments as mentioned previously.¹⁶ After obtaining blood from the tails using heparinized microhematocrit tubes, serum glucose of all groups was measured weekly prior to the beginning of the aqueous-extract treatment and on specified days during the treatment. Two weeks after streptozocin treatment, the serum glucose level of the diabetic rats reached 24-25 mM. One group of the streptozocin-induced hyperglycemic rats (experimental diabetic rats) received 1 mL aliquots of the aqueous extract twice a day (8:30 AM and 1:30 PM) through a gavage for a period of 12 days. The other diabetic group (diabetic control rats) had only the ad lib feeding schedule. A group of normoglycemic animals (normal experimental rats) received 1 mL aliquots of the aqueous extract once a day (8:30 AM) for a period of 10 days and the second normoglycemic group (normal control rats) followed only the ad lib feeding protocol. Since normoglycemic rats which received 1 mL aliquots of the extract twice daily became extremely hypoglycemic and died, it was decided to treat normoglycemic animals with only 1 mL aliquot of the extract once daily.

Serum glucose determination was performed on days 0 (just prior to aqueous-extract treatment), 1, 4, 8 and 12 in diabetic rats and on days 0, 2, 6 and 10 in normoglycemic animals after the intra- esophageal feeding of the extract to the experimental groups in the morning.

After 10 days of the aqueous extract treatment of the normoglycemic animals and 12 days after the extract treatment of the streptozocin-induced hyperglycemic rats, the animals were sacrificed and a portion of the livers were immediately placed in buffered 10% formalin. Two to three 5-micron-thick sections of the liver were prepared and stained with hematoxylin and eosin. The sections were studied by a pathologist who was not aware of the experimental treatments.

Table 1. Effects of aqueous extract of Teucrium polium on serum glucose levels of normoglycemic rats.

Table 2. Effects of aqueous extract of Teucrium polium on serum glucose levels of streptozocin-induced hyperglycemic rats.

The effects of the aqueous extract of the aerial parts of T. polium on serum glucose levels of normoglycemic and streptozocin-induced diabetic rats are shown in Tables 1 and 2, respectively. As shown in Table 1, intra-esophageal administration of 1 mL of the extract for 10 days had no significant effect on serum glucose level of the normoglycemic animals. However, twice daily treatment of the diabetic rats with 1 mL aliquots of the extract resulted in a highly significant reduction (p<0.001) in serum glucose level after 24h; the level reached those of the normoglycemic animals after 8 days of extract treatment (Table 2).

Figure 1 represents a typical normal hepatic lobule with a central vein and a portal tract. Liver sections from T. polium-treated rats showed marked hydropic change of the cytoplasm in one- third to two-third of the areas of the liver lobules (Figure 2). This type of change was mostly seen in

Figure 1. Typical normal hepatic lobule with a central vein (C) and portal tract (P). Original magnification x 200.

the perivenular and midzonal areas. Isolated hepatocyte necrosis (apoptopic bodies) was also easily observed especially in the perivenular zone (Figure 3). There was no significant inflammatory cell infiltrate in the portal as well as the injured areas, but Kupffer cells increased in number. Fatty changes were absent. The individual hepatocytes showed vesicular nuclei, nuclear enlargement with anisonucleosis and prominence of 1 or 2 nucleoli, suggestive of regenerative changes (Figure 3).

The histopathological changes of the liver (Figures 2 and 3) agree with the study of Mattei, et al⁵ who reported massive hepatocyte necrosis predominantly in the centrilobular areas of the liver in a patient with severe acute liver failure after T. polium consumption. Loeper, et al⁴ have also shown mid-zonal and centrilobular necrosis after 24 h in mice fed with 2.5 g of T. chamaedrys lyophilisate per kilogram body weight. Several cases of hepatitis due to T. chamaedrys have also been reported in France³. In this study, we observed midzonal and perivenular necrosis and hydropic changes in these two areas were also observed. Absence of inflammatory cells and the presence of scattered individual hepatocyte necrosis, suggests that cell injury had occurred by the apoptopic process following direct and rapid hepatotoxic damage passing through cellular swelling. Presence of cell activity i.e. nuclear enlargement and prominent nucleoli may demonstrate regenerative capacity and reversibility of the injurious process.

Figure 3. Histopathological changes of the diabetic liver treated with T. polium. Apoptotic bodies (A), increased Kupffer cells (K) and regenerative cells (R) are observed. Original magnification x 400.

In conclusion, we should emphasize that although the aqueous extract of T. polium aerial parts has potent hypoglycemic effects in experimental animals, it is strongly recommended that its use will be legislated against due to its proven hepatotoxic effects in both animals and humans.

Attempts should be made to evaluate the hypoglycemic effects of the organic and the inorganic constituents of this plant with the hope of separating the hypoglycemic factor(s) from the hepatotoxic ones.