Proliferative effect of aqueous extract of Hyptis fructicosa on liver regeneration after partial hepatectomy in rats

Efeito proliferativo do extrato aquoso da Hyptis fructicosa na regeneração hepática após hepatectomia parcial em ratos

12 – ORIGINAL ARTICLE

EFFECT OF DRUGS

Proliferative effect of aqueous extract of Hyptis fructicosa on liver regeneration after partial hepatectomy in rats¹

Efeito proliferativo do extrato aquoso da Hyptis fructicosa na regeneração hepática após hepatectomia parcial em ratos

Sônia Oliveira Lima^I; Luciano da Costa Viana^II; Fábio Rafael Teixeira de Santana^III; Ségio Zucoloto^IV; Ricardo Luiz de Albuquerque Junior^I; Margarete Zanardo Gomes^I

^IPhD, Full Professor, LMBE, ITP, Tiradentes University, Sergipe, Brazil. Conception and outlining of the study, responsible for intellectual and scientific contains of the article, acquisition and interpretation of data, manuscript writing, statistical analysis, responsible for English language

^IIFellow Master degree, Department of Morfology and Structural Biology (LMBA), Postgraduate Program in Health and Environment, Tiradentes University, Sergipe, Brazil. Acquisition and interpretation of data, manuscript writing, critical revision

^IIIMD, Postgraduate student, Department of Medicine, Federal University of Sergipe, Brazil. Statistical analysis, manuscript writing, critical revision

^IVPhD, Full Professor, Department of Pathology, Faculty of Medicine of Ribeirao Preto, Sao Paulo University, Brazil. Statistical analysis, analysis of specimens, manuscript writing, critical revision

^{Correspondece}

ABSTRACT

PURPOSE: To evaluate the effect of aqueous extract of Hyptis fructicosa on hepatic regeneration after partial hepatectomy in rats.

METHODS: Sixteen rats were divided in two groups: C (Control Group) and HF (Whose rats received aqueous extract of Hyptis fructicosa during 4 days using the dose of 100 mg/kg/day). On the consecutive day of this treatment, the animals of both groups underwent hepatectomy of about 67% of liver. Twenty four hours later, they were sacrificed, and the remaining mass of liver was removed and prepared to be studied through the PCNA immunohistochemical technique.

RESULTS: The liver regeneration index of HF group was 53.56 ± 18.91%, while in C group was 21.12 ± 8.29% (p=0.0003).

CONCLUSION: These results show that the administration of aqueous extract of Hyptis fructicosa using the dose of 100mg/kg/day increased the hepatocyte proliferation in the group HF.

Key words: Plants, Medicinal. Hepatectomy. Proliferating Cell Nuclear Antigen. Liver Regeneration. Hyptis. Rats.

RESUMO

OBJETIVO: Avaliar o efeito do extrato aquoso da Hyptis fructicosa sobre a regeneração hepática após hepatectomia parcial em ratos.

MÉTODOS: Dezesseis ratos foram divididos em dois grupos: C (grupo controle) e HF (ratos que receberam o extrato aquoso da Hyptis fructicosa durante quatro dias na dose de 100mg/kg/dia). No dia consecutivo deste tratamento, os animais de ambos os grupos foram submetidos a hepatectomia de aproximadamente 67% do fígado. Vinte e quatro horas depois, eles foram sacrificados, e que a massa restante do fígado foi retirado e preparado para ser estudado através da técnica de imuno-histoquímica PCNA.

RESULTADOS: O índice de regeneração hepática do grupo HF foi 53,56 ± 18,91%, enquanto no grupo C foi de 21,12 ± 8,29% (p=0,0003).

CONCLUSÃO: Estes resultados mostram que a administração do extrato aquoso da Hyptis fructicosa na dose de 100mg/kg/dia aumentou a proliferação de hepatócitos no grupo HF.

Descritores: Plantas Medicinais. Hepatectomia. Antígeno Nuclear de Célula em Proliferação. Regeneração Hepática. Hyptis. Ratos.

Introduction

Hyptis fructicosa Salzm ex Benth is a plant from the Lamiaceaea family¹ and is popularly known in Brazil as "Alecrim do Campo" or "Alecrim de Tabuleiro". This specie is about 0.5-1.5 m high and its leaves are aromatic, rhomboid and simple, and its flowers are clustered into axillary inflorescences, hermaphrodite and pentamer. It is popularly used to treat pain. Besides, it has been shown antinociceptive effect in mice². Two quinoids were isolated, from this plant, with antimicrobial and antineoplastic effect against Gram-positive microorganisms and mouse Erlich carcinoma studies, respectively³.

Studies using different species from the same gender were performed. It has been shown that the Hyptis suaveolens has positive effect on healing wounds⁴. The Hyptis ovalifolia has presented antimicrobial effect⁵. Also, the Hyptis pectinata has enhanced liver regeneration^6-8. However no article about the effect of Hyptis fructicosa on liver regeneration was found.

Considering that there are no studies defining the effects of this plant on liver, this research aimed at assessing the effects of Hyptis fructicosa leaves aqueous extract on liver regeneration after 67% partial hepatectomy in rats.

Methods

Hyptis fruticosa (Salzm. ex Benth.) Lamiaceae aerial parts were collected in September 2010, at "Feijão village" (10º56´S, 37º05´W), state of Sergipe, Brazil. The species was identified and authenticated by Dr. Adauto Souza Ribeiro. A voucher specimen of the plant (number ASE 01137) was deposited in the Herbarium of the Universidade Federal de Sergipe (UFS), Brazil. Its leaves were dried in an oven with air renewal and circulation (model MA-037) at 37°C until complete dehydration. Dried Hyptis fructicosa leaves were triturated in a blender until a finely granulated powder was obtained. The extract was obtained from this powder (100g) by adding distilled water (3:10, w/v) under constant shaking for 4h at 35^◦C, followed by filtration (pH 6.0). The filtrate was lyophilized (aqueous extract) and stored at 5^◦C. At the time of use, the extract was resuspended in distilled water at the desired concentrations.

The study was carried out on 16 male Wistar albino rats (300 to 450g). They were fed with a standard pellet diet and water ad libitum and kept at biotherium under natural light-dark cycle and environment temperature and humidity. All rats received humane care and the study protocol complied with the institution guidelines. All animals were randomly assigned to two groups, which consisted of eight rats each: CG, Control Group (n=8), whose rats received water daily for 4 days and HF, whose rats received aqueous extract of Hyptis fructicosa (HF group) during 4 days using the dose of 100 mg/kg/day. This dose showed no acute toxicity². On the consecutive day of this treatment, the animals of both groups underwent hepatectomy of about 67% of liver.

Surgical procedure

The operations were always performed at the same time in order to avoid the influence of the changes in circadian cycle. All operations were performed under ether anesthesia. In order to improve anesthesia performance and prevent problems such as air pollution, unstable anesthesia level and anesthetic wastefulness, an ether vapourizator for little rodents was used^9,10. The median and left lateral lobes of the liver (corresponding to 67% of the organ) were excised¹¹. After surgery, the rats were on a normal diet for the whole experimental period. Twenty four hours later, they underwent a new operation to remove the remaining liver. Then they were sacrificed.

Liver regeneration analysis

Liver regeneration was evaluated by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) using monoclonal primary anti-PCNA antibody (PC-10; DAKO A/S, Glostrup, Denmark) on formalin-fixed and paraffin-embedded liver tissues. Sections were cut at 4 μm, mounted on poly- L-lysine-coated glass slides, deparaffinized, rehydrated in an increasing alcohol series, placed in phosphate-buffered saline (PBS), and treated with 2% hydrogen peroxide in methanol for 15 min to block endogenous peroxidase activity. This was followed by incubation with PC-10 monoclonal primary antibody, diluted 1:40 in PBS for 120 min at 25^◦C. The sections were then incubated for 35 min with a biotinylated horse anti-mouse immunoglobulin. The reaction product was detected with an avidin–biotin–peroxidase complex and diaminobenzidine was used as a chromogen substrate. Positive and negative controls were used to assess and control the staining procedure. Sections were examined blindly at high power (400×), and 20 fields were chosen at random. Nuclear labeling indexes for PCNA (positive nuclei/total number of counted nuclei) were determined by evaluation of at least 1000 hepatocyte nuclei, as previously described^12,13. The specimens were processed by a pathologist who did not know which group he belonged to the animal.

Statistical analysis of data

All data are expressed as mean ± standard deviation. Statistical comparisons among the groups were performed by Mann-Whitney test. Probability values lower than 0.05 were considered to be statistically significant.

Ethic

This research was approved by the Ethic Committee in Researches using Animals from Universidade Federal de Sergipe – UFS, with the protocol number 36/2005.

Results

All data presented below were assessed 24h after 67% partial hepatectomy. The plant extract had been administered daily during 4 days, using the dose of 100 mg/kg of Hyptis fructicosa leaves aqueous extract, as previously explained.

Table 1 shows the individual results of liver regeneration index by each rat of both groups which was assessed by the proliferating cell nuclear antigen (PCNA) using monoclonal primary anti-PCNA antibody. Figure 1, shows the liver regeneration index in group HF was 53.56% ± 18.91, significantly increased compared to control group that presented index of 21.12 % ± 8.29 (p= 0.0003).

Discussion

There are several studies intended to comprehend the regeneration mechanism. It is known that some substances affect this process of regeneration. One study showed that the immunosuppressant tacrolimus has stimulatory effect on the process of liver regeneration after partial hepatectomy in rats¹⁴. An important number of growing factors can stimulate hepatoyte DNA synthesis, on primary culture, as HGF (hepatocyte growth factor), TGF-α (transforming growth factor- α) and EGF (epidermal growth factor). Some hormones participate on the liver regeneration process. The Norepinefrin acts on adrenegic α-1 receptors and amplifies the mitogenic signals of EGF and HGF¹⁵. The estradiol hexahydrobenzoate also promotes liver regeneration in rats submitted to a 70% hepatectomy¹⁶. The insulin together with glucagon stimulates the hepatocyte regeneration either in vitro¹⁷ or in vivo^18,19. The α2b interferon inhibits the hepatocyte proliferation when administered after 2 or 12h of the hepatectomy²⁰.

Also, anatomical factors can interfere. For instance, the hepatic branch of left vagus is important for the maintenance of the regenerative ability of liver, and the hepatic vagotomy promotes a delay in DNA synthesis^21,22. Partial portal vein arterialization (PPVA) is a method of iatrogenic promotion of liver regeneration in the setting of acute liver failure treatment²³. The partial occlusion of hepatic venous drainage in rats subjected to partial hepatectomy prolongs the proliferation of liver cells when compared to animals with veins of normal caliber²⁴. These data shows that liver regeneration is a complex phenomenon affected by different factors.

In our study the aqueous extract of Hyptis fructicosa augmented the liver regeneration in about 2.5 times compared with the control group. Former studies presented similar results using the extract of another plant, the Hyptis pectinata^6-8,25. As these plants belong to the same gender, it is possible that both plants share factors that can stimulate the liver regeneration, although the mechanism of this effect is yet unknown. As the Hyptis fucticosa is a phytotherapic, it may contain different substances which perhaps produce different effects. For instance, studies show that it has either antimicrobial effect against Gram-positive microorganisms and antineoplastic against mouse Erlich carcinoma²⁶. Another study showed that Hyptis fucticosa has anti-inflammatory and antioxidant²⁷.

A study presented an antinociceptive effect of this plant². This effect is similar to the effect presented by the Hyptis pectinata in others studies²⁸. As the antinociceptive effect of Hytpis fructicosa was reversed by naloxone, it also has been suggested that this may involve the activation of opiod receptors². Another study shows that the liver of rats with cholestasis express the preproenkephalin mRNA, which codes for the endogenous opioid peptide Met-enkephalin. Furthermore, Met-enkephalin immunoreactivity is detected in hepatocytes and in proliferating bile ductules in the cholestatic rat liver. This suggests that the opiods may have some effect on cholestatic liver and participate on liver regeneration²⁹. Thus, it is plausible that the Hyptis fructicosa effects on liver may be related with the activation of opioid receptors.

In contrast, the diterpenoid Horminone, a substance found in Hyptis fructicosa plants²⁶, was suggested to be toxic to the liver in rats³⁰. Other substances found in this plant are the taxodione and hyptol²⁶, however no publication about the effects of these substances on liver cells was found.

As there are few publications about the Hyptis fructicosa and the mechanism of liver regeneration may be influenced by different factors, new studies are necessary in order to isolate the substances presents and elucidate the mechanism which this plant enhance the liver regeneration.

Conclusion

The present study shows the aqueous extract of Hyptis fructicosa leaves at dose of 100mg/kg/day can stimulate the hepatic regeneration in rats.

Acknowledgment

The authors are grateful with Professor Lauro Xavier Filho for the support during the execution of this research.

Received: August 25, 2011

Review: October 20, 2011

Accepted: November 21, 2011

Conflict of interest: none

Financial source: none

• 1. Almeida CFCBR, Albuquerque UP. Check-list of the Family Lamiaceae in Pernambuco, Brazil. Braz Arch Biol Technol. 2002;45(3):343-53.
• 2. Silva ABL, Dias KS, Marques MS, Menezes IAC, Santos TC, Mello ICM, Lisboa ACCD, Cavalcanti SCH, Marçal RM, Antoniolli AR. Avaliação do efeito antinociceptivo e da toxicidade aguda do extrato aquoso da Hyptis fruticosa Salmz. ex Benth. Rev Bras Farmacogn. 2006;16(4):475-9.
• 3. Araújo MD, Cavalcanti MD, Méllo JF, D'Albuquerque IL, Lima OG, Monache FD, Maciel GM. Antimicrobial substances of superior plants. 45. Primary observations of 2 new quinoids isolated from Hyptis fructicosa Salzm ex Benth (Labiatae). Rev Inst Antibiot. 1974;14 (1-2):101-4.
• 4. Shirwaikar A, Shenoy R, Udupa AL, Shetty S. Wound healing property of ethanolic extract of leaves of Hyptis suaveolens with supportive role of antioxidant enzymes. Indian J Exp Biol. 2003;41(3):238-41.
• 5. Souza LKH, Oliveira CMA de, Ferri PH. Antimicrobial activity of Hyptis ovalifolia towards dermatophytes. Mem Inst Oswaldo Cruz. 2003;98(7):963-5.
• 6. Lima SO, Silva RL, Melo GB, Antoniolli AR. Efeito do extrato aquoso da Hyptis pectinata sobre a proliferação de hepatócitos após hepatectomia parcial. Acta Cir Bras. 2002;17(3):101-5.
• 7. Lima SO, Melo GB, Silva RL, Antoniolli AR. Efeitos do extrato aquoso da Hyptis pectinata sobre a regeneração hepática após hapatectomia parcial de 70%: Resultados Preliminares. Acta Cir Bras. 2001;16(1):13-5.
• 8. Melo GB, Melo VA, Lima SO, Antoniolli AR. Enhament of liver regeneration by the association of Hyptis pectinata with laser therapy. Dig Dis and Sci. 2005;50(5):949-54.
• 9. Souza F, Paes AMA, Alves HFP, Siroti FJ, Mathias PCF. Vaporizador de éter para cirurgia experimental em ratos. Acta Cir Bras. 1997;12(4):270-2.
• 10. Brito MVH, Brito NMB, Almeida AJ, Santos MRLC. Vaporizador artesanal de éter para cirurgia experimental em pequeno roedores. Acta Cir Bras. 1998;13(1):3-7.
• 11. Higgins GM, Anderson RM. Experimental pathology of the liver: restoration of liver of the white rat following partial surgical removal. Arch Pathol. 1931;12:186-202.
• 12. Ramalho FS, Ramalho LN, Castro-e-Silva O Jr, Zucoloto S, Corrêa FM. Effect of angiotensin-converting enzyme inhibitors on liver regeneration in rats. Hepatogastroenterology. 2002;49(47):134751.
• 13. Castro-e-Silva O Jr, Zucoloto S, Menegazzo LA, Granato RG, Marcassa LG, Bagnato VS. Laser enhancement in hepatic regeneration for partially hepatectomized rats. Lasers Surg Med. 2001;29(1):737.
• 14. Gama Filho O, Toderke EL, Baretta GAP, Sakamoto DG, Agulham MA, Tambara EM, Tambara EM, Matias JEF. Imunossupressão com tacrolimus favorece a regeneração hepática induzida por hepatectomia ampla em ratos. Rev Col Bras Cir. 2010;37(3):218-25.
• 15. Michalopoulos GK, Defrances MC. Liver Regeneration. Science. 1997;276:60-6.
• 16. Biondo-Simões MLP, Erdmann TR, Ioshii SO, Matias JEF, Calixto HLG, Schebelski DJ. The influence of estrogen on liver regeneration: an experimental study in rats. Acta Cir Bras. 2009;24 (1):3-6.
• 17. Friedman DL, Claus TH, Pilkis SJ, Pine GE. Hormonal regulation of DNA synthesis in primary cultures of adult rat hepatocytes: action of glucagon. Exp Cell Res. 1981;135(2):283-90.
• 18. Bucher NLR, Swaffield MN. Regulation of hepatic regeneration in rats bysynergistic action of insulin and glucagon. Proc Natl Acad Sci. 1975;72:1157-60.
• 19. Hwang TL, Chen MF, Chen TJ. Augmentation of liver regeneration with glucagon after partial hepatectomy in rats. J Formos Med Assoc. 1993;92:725-8.
• 20. Theocharis SE, Margeli AP, Skaltsas SD, Skopelitou AS, Mykoniatis MG, Kittas CN. Effect of interferon-a 2b administration on rat liver regeneration after partial hepatectomy. Dig Dis Sci. 1997;42(9):1981-6.
• 21. Lima SO, Ceneviva R, Martinelli ALC, Kajiwara JK, Zucoloto S, Castro-e-Silva O Jr. Role of the hepatic branch of the left vagus nerve in DNA synthesis by hepatocytes of rats subjected to partial hepatectomy. Res Surg. 1993;5(3):181-3.
• 22. Lima SO, Ceneviva R, Martinelli ALC, Zucoloto S. Truncal vagotomy influences on hepatocytes DNA synthesis folowing partial hepatectomy. Dis Dis Sci. 1986;31:397.
• 23. Nardo B, Caraceni P, Montalti R, Puviani L, Bertelli R, Beltempo P, Pacilè V, Rossi C, Gaiani S, Grigioni W, Bernardi M, Martinelli G, Cavallari A. Portal vein arterialization: a new surgical option against acute liver failure? Transplant Proc. 2005;37:2544-6.
• 24. Aguiar LRF, Nassif PAN, Ribas CAPM, Nascimento MM, Wiederker JC, Pachnicki JPA, Stieven-Filho E. Regeneração do fígado de ratos após oclusão parcial da drenagem venosa hepática. ABCD Arq Bras Cir Dig. 2009;22(2):89-95.
• 25. Melo GB, Silva RL, Melo VA, Antoniolli AR, Michellone PRT, Zucoloto S. Proliferative effect of the aqueous extract of Hyptis pctinata on liver regeneration after partial hepatectomy in rats. Acta Cir Bras. 2006;21(Suppl 1):S33-6.
• 26. Falcão DQ, Menezes FS. The Hyptis genus: an ethnopharmacological and chemical review. Rev Bras Farmaco. 2003;84(3):69-74.
• 27. Andrade AM, Oliveira JPR, Santos ALLM, Franco CRP, Antoniolli AR, Estevam CS, Thomazzi SM. Preliminary study on the anti-inflammatory and antioxidant activities of the leave extract of Hyptis fruticosa Salzm. ex Benth., Lamiaceae. Rev Bras Farmacogn. 2010;20(6):962-8.
• 28. Bispo MD, Mourao RH, Franzotti EM, Bomfi MKB, Arrigoni-Blank MF, Moreno MP, Marchioro M, Antoniolli AR. Antinociceptive and antiedematogenic effects of the aqueous extract of Hyptis pectinata leaves inexperimental animals. J Ethnopharmacol. 2001;76:81-6.
• 29. Nicoll J, Axiotis CA, Bergasa NV. The delta opioid receptor 1 is expressed by proliferating bile ductules in rats with cholestasis: implications for the study of liver regeneration and malignant transformation of biliary epithelium. Med Hypotheses. 2005;65(6):1099-105.
• 30. Ferreira R, Candeias F, Simões F, Nascimento J, Morais, JC. Effects of horminone on liver mixed function mono-oxygenases and glutathione enzyme activities of Wistar rat. J Ethnopharmacol. 1997;58(1):21-30.

1

Research performed at Laboratory of Morphology and Structural Biology (LMBE), ITP (Research and Technology Institute), Tiradentes University, Sergipe, Brazil.

Publication Dates

• Publication in this collection
  19 Dec 2011
• Date of issue
  Jan 2012

History

• Received
  25 Aug 2011
• Accepted
  21 Nov 2011
• Reviewed
  20 Oct 2011