RINGKASAN
POTENSI EKSTRAK DAUN MIANA (Coleus scutellarioides (L) Benth) SEBAGAI IMUNOMODULATOR PADA TIKUS MODEL
YANG TERINFEKSI Mycobacterium tuberculosis
Pemanfaatan Ekstrak Daun Miana untuk Pencegahan dan Pengobatan Tuberkulosis
Tuberkulosis adalah penyakit infeksi yang disebabkan oleh
Mycobacterium tuberculosis. Penyakit tuberkulosis dapat menulari siapa saja yang
berada di lingkungan penderita. Seseorang dengan kondisi imunitas tubuh yang baik, akan terhindar dari penyakit tuberkulosis, meskipun telah terpapar. Salah satu mekanisme imunitas dari tubuh terhadap penyakit tuberkulosis adalah fagositosis oleh makrofag. Aktivasi makrofag berfungsi meningkatkan fagositosis, meningkatkan destruksi mikroorganisma, meningkatkan kemotaksis, berperan sebagai antigen presenting cell, meningkatkan sekresi enzim dan substansi biologis. Peningkatan kapasitas fagositosis oleh sel-sel fagosit dan aktivitas fagositosis oleh makrofag, antara lain dipengaruhi oleh sitokin seperti interferon (IFN) dan tumor necrosis factor (TNF). Peningkatan imunitas bagi penderita tuberkulosis sangat penting, sehingga penderita tuberkulosis yang sedang menjalani pengobatan dengan strategi DOTS juga diberikan obat atau sediaan farmasi yang bersifat imunostimulan. Zat aktif yang terkandung dalam tanaman seperti flavonoid dan minyak atsiri merupakan komponen penting dalam menunjang imunitas tubuh karena dapat meningkatkan proliferasi limfosit.
Salah satu tanaman yang digunakan oleh masyarakat Toraja di Sulawesi Selatan untuk pengobatan tuberkulosis adalah miana (Coleus scutellarioides, (L) Benth). Survei (2013) mununjukkan sejumlah 85.71% dari penderita yang menggunakan obat tradisional memilih daun miana sebagai komplemen dalam pengobatan tuberkulosis. Secara in vitro ekstrak daun miana telah terbukti sebagai antibakteri. Meskipun telah digunakan secara empiris oleh suku Toraja untuk pengobatan tuberkulosis tetapi pembuktian ilmiah secara in vivo belum ada. Sehingga daun miana sangat potensial untuk diteliti dan dikembangkan mengingat kasus tuberkulosis di Indonesia masih sangat tinggi. Tujuan penelitian ini adalah membuktikan potensi ekstrak daun miana sebagai imunomodulator terhadap pencegahan dan pengobatan tuberkulosis, serta sebagai komplemen obat antituberkulosis.
pengobatan placebo, rifampisin, EDM dan CRM (Campuran rifampisin dan EDM). Kelompok 7, 8 dan 9 adalah kelompok pencegahan yaitu hewan yang diberi EDM sebelum diinfeksi dan setelah sakit diberikan placebo, EDM dan CRM. Hewan sakit karena diinfeksi dengan Mycobacterium tuberculosis strain H37Rv. Dosis EDM digunakan 510 mg/kg BB tikus. Dosis rifampisin 45 mg/kg BB tikus. Masa infeksi adalah 30 hari dan lama perlakuan 21 hari. Parameter imunomodulator yang diuji dalam penelitian ini adalah proliferasi limfosit-T, sel-T CD4, IFN-γ, TNF-α dan koloni Mycobacterium tuberculosis (M. tb). Jumlah limfosit-T dan jumlah sel-T CD4 diukur dengan metode flowcytometry, kadar IFN-γ dan kadar TNF-α diukur dengan metode ELISA, jumlah koloni Mycobacterium tuberculosis (M. tb) dihitung berdasarkan hasil kultur paru tikus pada media Lowenstein Jensen. Data pengamatan dianalisis statistik non parametric Kruskal-Wallis dan Mann-Whitney kemudian analisis hubungan dengan Amos.
Hasil penelitian menunjukkan bahwa pemberian ekstrak daun miana (EDM) berpengaruh terhadap peningkatan proliferasi limfosit-T. Berdasarkan analisis Mann Whitney, secara umum ditemukan bahwa ada perbedaan jumlah limfosit-T antar kelompok meskipun beberapa kelompok tidak signifikan, tetapi semua kelompok berbeda signifikan terhadap kontrol hewan sehat (kelompok 1). Hasil penelitian ini membuktikan bahwa EDM berpengaruh terhadap peningkatan proliferasi limfosit-T sehingga dapat berfungsi sebagai imunomodulator yang berperan meningkatkan imunitas (imunostimulan). Perbandingan antar kelompok perlakuan menunjukkan hasil bahwa peningkatan jumlah limfosit-T yang terbaik ditemukan pada pemberian komplemen EDM dan obat anti tuberkulosis yaitu kelompok 9 (diberikan EDM untuk pencegahan dan CRM untuk pengobatan) dan kelompok 6 yaitu kelompok yang diberikan CRM sebagai pengobatan.
Ditemukan bahwa EDM berpengaruh terhadap peningkatan jumlah sel-T CD4. Pada penelitian ini semua perlakuan memberikan hasil yang berbeda antar kelompok, kecuali kelompok 5-7 tidak signifikan yaitu pemberian EDM sebagai pengobatan tidak berbeda dengan pemberian EDM sebagai pencegahan. Semua perlakuan terhadap hewan uji berpengaruh meningkatkan sel-T CD4 dibandingkan kontrol hewan sehat. Hal ini menunjukkan bahwa EDM dapat berfungsi sebagai imunomodulator yaitu imunostimulan dalam pengobatan tuberkulosis dan berperan sebagai imunoregulator dalam pencegahan tuberkulosis. Perbandingan peningkatan jumlah sel-T CD4 pada setiap kelompok perlakuan dalam penelitian ini menunjukkan hasil bahwa yang terbaik ditemukan pada kelompok 9 dan kelompok 6.
IFN-γ menunjukkan bahwa hasil yang terbaik ditemukan pada kelompok 9 dan kelompok 6.
Data penelitian yang dianalisis secara statistik memberikan hasil bahwa EDM berpengaruh terhadap peningkatan kadar sitokin TNF-α dan analisis selanjutnya menunjukkan tidak ada perbedaan kadar sitokin TNF-α antara kelompok 2-6 dan 6-9. Hal ini menunjukkan bahwa EDM sebagai imunomodulator dapat meningkatkan imunitas dalam pengobatan tuberkulosis. Data ini membuktikan bahwa EDM berfungsi sebagai komplemen dalam pengobatan tuberkulosis. Perbandingan peningkatan TNF-α menunjukkan bahwa hasil yang terbaik ditemukan pada kelompok 9 dan kelompok 6.
Hasil penelitian menunjukkan bahwa EDM berpengaruh terhadap penurunan jumlah koloni M. tb. Data penelitian menunjukkan tidak ada pertumbuhan koloni M. tb pada perlakuan kelompok 1,2,6 dan 9 yaitu kelompok hewan sehat dan kelompok hewan sakit yang diberi pengobatan CRM. Analisis selanjutnya menunjukkan semua perlakuan lainnya ada perbedaan jumlah koloni M. tb. Hal ini menunjukkan tidak ada pertumbuhan koloni M. tb pada hasil kultur paru tikus sakit yang mendapat pengobatan CRM dan fungsi EDM sebagai imunomodulator dapat meningkatkan imunitas dalam pengobatan tuberkulosis. Data ini membuktikan bahwa EDM berfungsi sebagai komplemen dalam pengobatan tuberkulosis.
Analisis hubungan antar variabel dengan Amos menunjukkan bahwa peningkatan proliferasi limfosit-T berhubungan signifikan dengan peningkatan jumlah sel-T CD4 tetapi tidak berhubungan langsung dengan penurunan jumlah koloni M. tb. Peningkatan jumlah sel-T CD4 berhubungan signifikan terhadap peningkatan kadar IFN-γ, peningkatan kadar TNF-α dan terhadap penurunan jumlah koloni M. tb. Peningkatan kadar IFN-γ berhubungan siginifikan terhadap penurunan jumlah koloni M. tb. Demikian pula peningkatan kadar TNF-α berhubungan siginifikan terhadap penurunan jumlah koloni M. tb.
Analisis hubungan antar variabel pada perlakuan pencegahan menunjukkan bahwa peningkatan proliferasi limfosit-T berhubungan signifikan terhadap peningkatan jumlah sel-T CD4 tetapi tidak berhubungan langsung terhadap penurunan jumlah koloni M. tb. Peningkatan jumlah sel-T CD4 berhubungan signifikan terhadap peningkatan kadar sitokin IFN-γ, kadar sitokin TNF-α tetapi tidak berhubungan langsung terhadap penurunan jumlah koloni M. tb. Peningkatan IFN-γ berhubungan siginifikan terhadap penurunan jumlah koloni M. tb, tetapi peningkatan TNF-α tidak berhubungan langsung terhadap penurunan jumlah koloni M. tb.
Analisis hubungan untuk perlakuan pengobatan menunjukkan bahwa peningkatan proliferasi limfosit-T tidak berhubungan langsung dengan peningkatan sel-T CD4 dan terhadap penurunan jumlah koloni M. tb. Peningkatan jumlah sel-T CD4 berhubungan signifikan terhadap peningkatan kadar IFN-γ dan kadar TNF-α tetapi tidak berhubungan langsung terhadap penurunan jumlah koloni M. tb. Selanjutnya peningkatan kadar IFN-γ dan peningkatan kadar TNF-α berhubungan siginifikan terhadap penurunan jumlah koloni M. tb.
TNF-α. EDM juga berpengaruh menurunkan jumlah koloni M. tb pada paru tikus yang terinfeksi tuberkulosis. EDM efektif sebagai komplemen dalam pengobatan tuberkulosis. Hubungan antar variabel menunjukkan proliferasi limfosit-T meningkatkan jumlah sel-T CD4 yang selanjutnya meningkatkan kadar IFN-γ dan kadar TNF-α yang kemudian menurunkan jumlah koloni M. tb. Hubungan pada kelompok pencegahan adalah proliferasi limfosit-T meningkatkan jumlah sel-T CD4 yang selanjutnya meningkatkan kadar IFN-γ dan kemudian menurunkan koloni M. tb. Hubungan pada kelompok pengobatan adalah sel-T CD4 meningkatkan kadar IFN-γ dan kadar TNF-α yang selanjutnya menurunkan jumlah koloni M. tb. Berdasarkan hasil penelitian dan kesimpulan maka disarankan untuk menggunakan EDM sebagai imunomodulator pada pencegahan tuberkulosis dan sebagai komplemen dalam pengobatan tuberkulosis serta melakukan penelitian lebih lanjut untuk mengetahui: Potensi EDM sebagai hepatoprotektor pada pemberian obat anti tuberkulosis dan potensi EDM pada perbaikan kerusakan paru akibat tuberkulosis.
SUMMARY
Potentiality of Miana (Coleus scutellariodes (L) Benth) Leaf Extracts as Immunomodulator on Rats Infected by Mycobacterium tuberculosis
(The Use of Miana Leaf Extracts as Tuberculosis Preventive and Curative)
Tuberculosis is a contagious disease caused by Mycobacterium tuberculosis. It may infect anybody around the patient. Individuals with a good immune condition may be resistant to tuberculosis although he/she has been intensely exposed to the patient. One of body immune mechanisms against tuberculosis is phagocytosis by macrophage. Macrophage activation functions to enhance phagocytosis, destruct microorganisms, and enhance chemotaxis, and acts as an antigen presenting cell to enhance enzymatic secretion and biological substances. Improvements on phagocytosis capacity by the phagocytes and phagocytosis activity by macrophage cells are affected by cytokine, such as Interferon (INF)
and Tumor Necrosis Factor (TNF). Improvement on body immune system is
essential for Tuberculosis patients. Therefore, medication and pharmaceutical preparations are given to Tuberculosis patients undergoing treatment based on DOTS strategy to stimulate their immune system. Active natural compounds (e.g. flavonoids and ethereal oil) are among the essential components in improving body immune system due to their effect to enhance lymphocytes proliferation.
One of herbs which is commonly used by Toraja community in South Sulawesi Province as a medical treatment on Tuberculosis is Miana (Coleus scutellarioides (L.) Benth). Based on a survey conducted in Toraja community in 2013, about 85.71% respondents used Miana leaf as a complementary medication to treat Tuberculosis. Based on in vitro examination, Miana leaf extracts (MLE) has been proven to possess an antibacterial function. Despite of it’s empirically use to treat Tuberculosis by Toraja community, in vivo examination to scientifically prove the potentiality of MLE as treatment on Tuberculosis has not been conducted. Considering the high rate of Tuberculosis in Indonesia, Miana is potentially studied and developed as treatment on Tuberculosis. Hence, this research was intended to prove the potentiality of MLE as an immunomodulator in preventing and curing Tuberculosis.
This research was considered as a pure-experimental research using male Wistar rats as treated animals. The rats were divided randomly into 9 treatment groups, each consisted of 10 rats. Treatment groups 1 and 2 were healthy (uninfected) rats treated using a placebo and MLE, respectively. Treatment groups 3, 4, 5, and 6 were curative groups consisting of infected rats treated using curative placebo, rifampicin (an anti-tuberculosis drug), MLE, and MIX compounds (mixture of MLE and rifampisin),respectively. Meanwhile, treatment groups 7, 8, and 9 were preventive groups which were treated using MLE before being artificially infected by Mycobacterium tuberculosis. After infection, these curative groups were treated using placebo, MLE, and MIX compounds, respectively. All the rats were infected by Mycobacterium tuberculosis strain
H37Rv. The dosage of MLE given to the rats was 510 mg/ kg BW and the dosage
30 days and treatment period was 21 days. Immunomodulator parameters examined in this research were T-lymphocytes proliferation, and CD4 T-cells, IFN-γ, and TNF-α, and Mycobacterium tuberculosis colonies formed. The numbers of T-lymphocytes and CD4 T-cells were measured based on flowcytometry method. IFN-γ and TNF-α contents were measured using ELISA, while the number of Mycobacterium tuberculosis colonies formed were measured based on examination on the rat lung tissue cultured on Lowenstein Jensen medium.
The findings of this research indicated that MLE treatment potentially enhanced T-lymphocytes proliferation. Mann Whitney tests indicated in general there were different numbers of T-lymphocytes among the treatment groups although the differences were insignificant. However, these numbers were very significantly compared to the control group (treatment group 1 consisting of healthy rats with placebo). This result suggested that MLE affected T-lymphocytes enhancement; thus it potentially functioned as immunostimulant to improve immune system. Based on comparison among the treatment groups of infected rats, it was found that T-lymphocytes enhancement was found on treatment group 9 (where preventive was given and the MIX compound was given as curative) and treatment group 6 (where the MIX compound was given as curative).
It was also found that MLE treatment had resulted on significant improvement on CD4 T-cell numbers, except for treatment groups 5-7 in which there was insignificant difference between MLE given as preventive and curative treatments. All treatment groups indicated significant improvements on CD4 T-cell numbers compared to the control group (group 1). This result indicated that MLE might function both as immunomodulator (i.e. immunostimulant) in curing tuberculosis and as immunoregulator in preventing tuberculosis. Based on the comparison among the treatment groups, of infected rats CD4 T-cell improvements were found on groups 6 and 9.
The findings of this research also demonstrated that MLE affected IFN-γ cytokine improvement. The results of Kruskal-Wallis data analysis indicated significant differences on IFN-γ contents among the treatment groups. Meanwhile, the result of Mann Whitney analysis suggested there were significant differences on IFN-γ contents among all the treatment groups, except for treatment groups 1 and 2 indicating both MLE and placebo did not have significant effects on the healthy rats with groups 2 whereas there was a significant improvement on the treatment groups’ IFN-γ contents compared to the control. This finding proved that Miana leaf extracts potentially functioned as an immunomodulator to improve immune system (as immunostimulant) in curing tuberculosis. The result of IFN-γ contents improvement comparison of infected rats indicated IFN-γ improvement was found on treatment groups 6 and 9.
healthy samples (control group) treated using Miana leaf extracts. This finding proved that Miana Leaf extracts was potentially used as a complementary medication functioned as immunomodulator in curing tuberculosis. The result of TNF-α contents improvement comparison of infectes rats indicated the highest TNF-α improvement was found on treatment groups 6 and 9.
MLE could also reduce the number of Mycobacterium tuberculosis colonies in the lung tissue of the treated rats. Based on the examination, there was no
Mycobacterium tuberculosis grown on treatment groups 1, 2, 6, and 9 (treatment
groups consisting of healthy rats and infected rats treated using MIX compounds). Further analysis on the data showed there were different numbers of Mycobacterium tuberculosis colonies formed among the treatment groups. This result also indicated that the number of Mycobacterium tuberculosis colonies formed on infected samples receiving the MIX compound treatment (group 6 and 9) was not significantly different from the healthy rats indicating that Miana leaf extracts as immunomodulator could improve immune system in curing tuberculosis thus it can be potentially used as a complementary medication in tuberculosis treatment.
The results of inter-variable correlation analysis suggested that T-lymphocytes proliferation improvement significantly increased the number of CD4 T-cells. However, T-lymphocytes proliferation had no effects on reducing
Mycobacterium tuberculosis colonies. The improvement on CD4 T-cells
significantly enhanced IFN-γ, and TNF-α contents, and reduced the number of Mycobacterium tuberculosis colonies formed in the lung tissue of the rats. The improvement on IFN-γ content could significantly reduce the number of
Mycobacterium tuberculosis colonies. Similarly, the improvement on TNF-α
content could significantly reduce the number of Mycobacterium tuberculosis colonies.
The results of inter-variable correlation analysis on preventive treatment groups indicated that the improvement on T-lymphocytes proliferation significantly increased the number of CD4 T-cells yet could not reduce the number of Mycobacterium tuberculosis colonies formed. The increasing numbers of CD4 T-cells significantly enhanced IFN-γ and TNF-α contents yet could not reduce the number of Mycobacterium tuberculosis colonies. The enhancement on IFN-γ significantly reduced the numbers of Mycobacterium tuberculosis but the enhanced TNF-α could not directly reduce the numbers of Mycobacterium tuberculosis colonies.
The results of inter-variable correlation analysis on curative treatment groups indicated that the improvement on T-lymphocytes proliferation did not directly increase the number of CD4 cells. The increasing numbers of CD4 T-cells significantly enhanced IFN-γ content and the increase of TNF-α content yet could not reduce the number of Mycobacterium tuberculosis colonies. Yet, the enhancements on IFN-γ content and TNF-α content could significantly reduce the number of Mycobacterium tuberculosis colonies.
reduce Mycobacterium tuberculosis colonies in rat lung infected by tuberculosis. The result of inter-variable correlation analysis indicates T-lymphocytes proliferation may improve the number of CD4 T-cells which in turn enhances IFN-γ and TNF-α contents, and reduce the number of Mycobacterium tuberculosis colonies formed. The result of inter-variable correlation on the preventive treatment groups indicates T-lymphocytes proliferation may improve the number of CD4 T-cells which in turn enhances IFN-γ and reduce Mycobacterium tuberculosis colonies. The result of inter-variable correlation analysis on the curative treatment indicates that CD4 T-cells may improve IFN-γ content and
TNF-α content which reduce Mycobacterium tuberculosis colonies. As
recommendations, further researchers are expected to conduct advanced researches to examine the potentiality of MLE as a hepatoprotector in anti-tuberculosis medication and its potentiality in recovering damaged lung cells caused by tuberculosis.
This research resulted in several new concepts: (1). EDM (C. scutellarioides (L) Benth) proved to be potentially as an immunomodulator in the prevention and treatment of tuberculosis through immunostimulatory mechanism by increasing the proliferation of T lymphocytes, CD4 T-cell count, increased levels of IFN-γ and TNF-α levels thereby reducing the number of colonies M. tb bacteria. (2). EDM (C. scutellarioides (L) Benth) as a complement in the treatment of tuberculosis rifampicin provide healing in mice proved the absence of growth of colonies of M. tb in rat lung culture results. (3). EDM (C. scutellarioides (L) Benth) provide the initial mechanism for defense against intracellular bacterial infections. (4). The potential of EDM (C. scutellarioides (L) Benth) as prevention of tuberculosis through the EDM - proliferation of T lymphocytes - CD4 T-cells - IFN-γ - M. tb. (5). The potential of EDM (C. scutellarioides (L) Benth) as a treatment for tuberculosis through the EDM - CD4 T-cells - IFN-γ - M. tb and EDM - CD4 T-cells - TNF-α - M. tb.
ABSTRAK
Penelitian bertujuan membuktikan potensi ekstrak daun miana (Coleus
scutellarioides (L) Benth) sebagai imunomodulator dan komplemen terhadap
tikus model yang terinfeksi Mycobacterium tuberculosis (M. tb). Sampel tikus putih Wistar jantan terbagi 9 kelompok, 4 kelompok mewakili pemanfaatan ekstrak daun miana (EDM) untuk pencegahan dan 5 kelompok untuk pengobatan tuberkulosis. Sampel diinfeksi M. tb strain H37Rv secara intra trakeal, diberikan perlakuan oral EDM, rifampisin, CRM (Campuran rifampisin dan EDM) dan placebo. Parameter imunomodulator yang diuji adalah jumlah limfosit-T dan jumlah sel-T CD4 diukur dengan metode flowcytometry, kadar IFN-γ dan kadar TNF-α diukur dengan metode ELISA serta jumlah koloni M. tb hasil kultur paru tikus pada media LJ.
Hasil penelitian menunjukkan EDM berpengaruh meningkatkan jumlah limfosit-T dan sel-T CD4, kadar IFN-γ dan TNF-α serta menurunkan jumlah koloni M. tb. Perlakuan EDM sebagai komplemen (CRM) menunjukan tidak ada pertumbuhan koloni M. tb pada paru tikus. Analisis hubungan dengan Amos menunjukkan proliferasi limfosit-T meningkatkan sel-T CD4. Sel-T CD4 meningkatkan IFN-γ, TNF-α dan menurunkan koloni M. tb. IFN-γ dan TNF-α menurunkan koloni M. tb.
Analisis hubungan untuk perlakuan pencegahan menunjukkan limfosit-T meningkatkan sel-T CD4. Sel-T CD4 meningkatkan IFN-γ dan TNF-α. IFN-γ menurunkan M. tb. Analisis hubungan pada perlakuan pengobatan menunjukkan sel-T CD4 meningkatkan IFN-γ dan TNF-α. IFN-γ dan TNF-α menurunkan koloni M. tb.
Kesimpulan EDM berpotensi sebagai imunomodulator dan komplemen dengan mekanisme meningkatkan jumlah limfosit-T, jumlah sel-T CD4, kadar IFN-γ, kadar TNF-α dan menurunkan jumlah koloni M. tb. Hubungan antar variabel menunjukkan Proliferasi limfosit-T meningkatkan sel-T CD4 kemudian meningkatkan IFN-γ dan TNF-α selanjutnya menurunkan koloni M. tb.
ABSTRACT
This research was conducted to prove the potency of Miana (Coleus scutellaroides (L) Benth) leaf extracts as immunomodulator and complement on rats infected by Mycobacterium tuberculosis (M. tb). The rats used in this research were male Wistars divided into 9 groups: 4 groups treated with Miana Leaf Extract (MLE) as the tuberculosis preventive substance and 5 groups treated with MLE as the tuberculosis curative substance. The rats were infected with M. tb strain H37Rv byintra-tracheal injection and then given orally either, MLE, anti-tuberculosis drugs (rifampicin ), MIX (mixture of Miana leaf extracts and anti-tuberculosis drugs) or placebo. Immunomodulator parameters examined in this research were the numbers of T-lymphocytes and CD4 T-cells (measured using flowcytometry method), IFN-γ and TNF-α contents (measured based on ELISA method), and the number of M. tb formed in rats lung tissue cultured on LJ medium.
The findings of this research indicated that MLE affected the increasing number of T-lymphocytes, CD4 T-cells, IFN-γ and TNF-α level, and reduced the number of the M. tb colonies in rats lung tissue. The result of AMOS (Analysis of Moment Structures) correlation analysis for either preventive or curative treatment indicated that the T-lymphocytes proliferation affected CD4 T-cells which in turn affected the IFN-γ and TNF-α level. Both of the IFN-γ and TNF-α at the end also affected the decrease of the M. tb colonies.
The result of correlation analysis on preventive treatment indicated that T-lymphocytes affected CD4 T-cells. CD4 T-cells affected IFN-γ and TNF-α level. IFN-γ level affected M. tb colonies. On the other hands, the result of correlation on curative treatment indicated that CD4 T-cells affected IFN-γ and TNF-α level. Eventually, IFN-γ and TNF-α would affect M. tb colonies.
Based on these findings, it can be concluded that MLE potentially functions as an immunomodulator through a mechanism of by means of improving the number of T-lymphocytes, CD4 T-cells, IFN-γ and TNF-α level and reducing the number of M. tb formed on lung tissues. Positive correlations among the variables indicated T-lymphocytes proliferation affected CD4 T-cells enhancement which in turn improved the level of IFN-γ and TNF-α and reduced the number of M. tb colonies.
