Diterpenes from ”Pini Resina” and their Preferential Cytotoxic Activity under Nutrient-Deprived Condition

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

In the course of our search for anticancer agents based on a novel anti-austerity strategy, we found that the 70 % EtOH extract of ”Pini Resina” showed 100 % preferential cytotoxicity at the concentration of 50 μg/mL. Further bioassay-guided fractionation and purification led to the isolation of 15 compounds including one new compound 7-oxo-13α-hydroxyabiet-8(14)-en-18-oic acid (1). Their structures were elucidated on the basis of spectroscopic analysis. Among the isolated compounds, methyl abieta-8,11,13-trien-18-oate (7) showed the most potent preferential cytotoxicity at 10 μg/mL under nutrient-deprived condition.

Pancreatic cancer is an aggressive disease with the lowest survival rate of all cancers at 5 years [1], [2]. It is largely resistant to conventional forms of treatment and the development of more effective treatment is urgently needed. Previously, we reported that certain pancreatic cancer cell lines such as PANC-1, AsPC-1, BxPC-1, and KP-3 showed remarkable tolerance against extreme nutrient starvation [3] and the elimination of this tolerance of cancer cells to nutrient starvation might serve as a novel approach for cancer therapy [3], [4], [5], [6]. In this regard, we screened 500 medicinal plant extracts used in Japanese ”Kampo” medicine for their preferential cytotoxicity against PANC-1 cancer cells under nutrient-deprived conditions. Among them, the 70 % EtOH extract of ”Pini Resina” showed 100 % cytotoxicity preferentially in nutrient-deprived medium (NDM) at the concentration of 50 μg/mL. Thus, bioassay-guided fractionation and isolation of an active compound were carried out.

”Pini Resina” or ”Pine Resin” is the natural resin obtained from conifers and is commercially produced from about 20 pine species in many developing countries [7]. Traditionally, ”Pine Resin” has been used to resolve inflammations, to relieve the symptoms of cough, and to treat urinary problems [8], [9]. In Japanese ”Kampo” medicine, ”Pini Resina” is used for the treatment of skin diseases, diabetes, and tuberculosis and as a pain killer [10]. In the present study, bioassay-guided fractionation and purification led to the isolation of 15 compounds including one new compound 7-oxo-13α-hydroxyabiet-8(14)-en-18-oic acid (1).

”Pini Resina” was extracted with 70 % EtOH and the EtOH extract was chromatographed on silica gel with a MeOH/CHCl₃ solvent system to give nine fractions. These fractions were examined for their preferential cytotoxic activity in NDM and the fractions 2 - 4 showed preferential cytotoxicity at 50 μg/mL. Thus, they were further separated to afford 15 diterpenes (1 - 15). The structures of known compounds were determined by comparisons of their spectral data with those in literature and were found to be 13,14-seco-13-oxoabiet-7-ene-14,18-dioic acid (2) [11], 8(14),15-pimaradien-18-oic acid (3) [12], 7-oxodehydroabietic acid (4) [13], 7-oxo-13β-hydroxyabiet-8(14)-en-18-oic acid (5) [14], methyl 7-oxoabieta-8,11,13-trien-18-oate (6) [15], methyl abieta-8,11,13-trien-18-oate (7) [16], 8(14)-podocarpen-7,13-dion-18-oic acid (8) [17], 8(14)-podocarpen-13-on-18-oic acid (9) [17], 12-hydroxydehydroabietic acid (10) [18], 13,14-seco-13,14-dioxoabiet-7-en-18-oic acid (11) [19], 8,11,13-abietatrien-18-oic acid (12) [16], 16-nor-15-oxodehydroabietic acid (13) [20], 8(14),15-pimaradien-18-ol (14) [12], and 12-oxoabieta-7,13-dien-18-oic acid (15) [21].

Compound 1 was isolated as colorless amorphous solid with [α]_D ²²: -27° (CHCl₃) and its molecular formula was determined to be C₂₀H₃₀O₄ by high resolution (HR)-EI-MS. The IR spectrum showed the presence of hydroxy (3600 cm^-1) and carbonyl (1690 cm^-1) groups. The ¹H-NMR spectrum of 1 displayed signals due to two tertiary methyls, two secondary methyls, six methylenes, an isolated olefinic proton, and three aliphatic methines (Table [1]). On the other hand, the ¹³C-NMR spectrum showed the signals of 20 carbons including those of a ketone carbonyl carbon (δ _C = 199.8), two olefinic carbons, and one oxygenated and two aliphatic quaternary carbons (Table [1]). These ¹H- and ¹³C-NMR spectral data, and its molecular formula, were similar to those of the known compound 7-oxo-13β-hydroxyabiet-8(14)-en-18-oic acid (5) [14], isolated from the same extract, except for the slight difference in the chemical shift values in their ¹H- and ¹³C-NMR spectra. Analysis of the ¹H-¹H COSY, HMQC, and HMBC spectra (Fig. [1] A) of 1 gave the same planar structure as 5, suggesting that 1 is a stereoisomer of 5. Furthermore, in the ROESY experiment, the correlations H₃-19/H₃-20, H-6β/H₃-20, H-6β/H₃-19, H-5/H-9, and H-1/H-9 (Fig. [1] B) indicated rings A and B to be trans-fused, with β-axial orientations of the C-19 and C-20 methyl groups, and α-axial orientations of H-5 and H-9, which is identical to the known compound 5. Thus, 1 and 5 should differ in the stereochemistry at C-13; i. e., the hydroxy group at C-13 in 1 should be α-oriented. As for ring C, the NOE enhancements between H₃-20 and H-12β (δ   = 1.41), H₃-16 and H-12α (δ = 2.16), H₃-17 and H-12α, H-9 and H-11α (δ = 1.39), H₃-16 and H-14, H₃-17 and H-14 were observed. The low-field shift of H-9 in 1 (δ _H = 2.22) compared to that in 5 (δ _H = 2.02) might be attributable to anisotropic deshielding by the α-oriented hydroxy group at C-13. From these data, 1 was determined to be 7-oxo-13α-hydroxyabiet-8(14)-en-18-oic acid.

Compound 2 was isolated as a colorless amorphous solid and a literature survey on 2 suggested it to be 13,14-seco-13-oxoabiet-7-en-14,18-dioic acid, previously reported as a semisynthetic reaction product of abietic acid [11]. In our present investigation, we isolated 2 as a natural product for the first time and assigned its ¹H- and ¹³C-NMR data (Table [1]) based on ¹H-¹H COSY, HMQC and HMBC spectral data.

The isolated compounds were tested for their preferential cytotoxic activity under nutrient deprived condition. Preferential cytotoxicity is defined as the death of cancer cells selectively in nutrient-deprived medium (NDM) with no cytotoxicity in ordinary nutrient-rich medium (DMEM) [4], [5], [6]. Among the tested diterpenes, compound 7 exhibited the most potent preferential cytotoxic activity at 10 μg/mL (Fig. [2]). Compound 3, 6, 12, 13, and 14 showed moderate preferential cytotoxicity at 50 μg/mL while 4, 5, 8, 10 and 11 exhibited weak preferential cytotoxicity at 200 μg/mL. Compounds 1 and 2 were inactive at the maximum concentration tested, i. e., >200 μg/mL (Fig. [1]S in the Supporting Information). From the observed activity data, it can be concluded that esterification of the C-18 carboxyl group significantly enhances the activity (7 > 12). Esterification of the acid group, basically, renders the compound less polar, which could be responsible for the better penetration of the compound across the lipid bilayer surrounding the cells. All the tested diterpenes with a ketone group at C-7 showed weaker activity than the corresponding C-7 methylene compound (6 < 7, 4 < 12, 8 < 9). Additionally, the hydroxy group at C-12 also decreased the activity (10 < 12). As compound 7 was an extremely minor compound, the observed cytotoxic activity in ”Pini Resina” extract might be attributable to the major compounds such as 3 and 12.

Materials and Methods

Optical rotations were recorded on a JASCO DIP-140 digital polarimeter. IR spectra were measured with a Shimadzu IR-408 spectrophotometer in CHCl₃ solution. NMR spectra were taken on a JEOL JNM-LA400 spectrometer in CDCl₃ solution with tetramethylsilane (TMS) as an internal standard, and chemical shifts are expressed in δ values. HR-EI-MS measurements were carried out on a JEOL JMS-700T spectrometer. Column chromatography was performed with BW-820MH silica gel (Fuji Silysia; Aichi, Japan). Analytical and preparative TLC were carried out on precoated silica gel 60F₂₅₄ or RP-18F₂₅₄ plates (Merck; Darmstadt, Germany; 0.25 or 0.50 mm thickness).

Commercial ”Pini Resina” imported from China was purchased from Alps Pharmaceutical Company (Gifu, Japan).

”Pini Resina” (90.0 g) was extracted with 70 % EtOH-H₂O (3 L, 24 h × 3) at room temperature to give 87.0 g of extract which was subjected to silica gel column chromatography (7 × 40 cm) with a MeOH/CHCl₃ (0 - 50 %) gradient system to give nine fractions: Fr. 1 (0.1 g), CHCl₃ eluate (1 L); Fr. 2 (14.2 g), CHCl₃ eluate (4 L); Fr. 3 (6.3 g), 5 % MeOH-CHCl₃ eluate (2 L); Fr. 4 (5.1 g), 8 % MeOH-CHCl₃ eluate (2 L); Fr. 5 (6.3 g), 10 % MeOH-CHCl₃ eluate (2 L); Fr. 6 (6.8 g), 15 % MeOH-CHCl₃ eluate (2 L); Fr. 7 (10.1 g), 20 % MeOH-CHCl₃ eluate (2.5 L); Fr. 8 (12.5 g), 30 % MeOH-CHCl₃ eluate (3 L); and Fr. 9 (18.2 g), 50 % MeOH-CHCl₃ eluate (3 L).

Fr. 2 was rechromatographed (4 × 30 cm) on silica gel with MeOH/CH₂Cl₂ (0 - 50 %), and the subfractions obtained were further separated by reversed-phase preparative TLC with MeCN/acetone/H₂O (1 : 1:1), to give the following compounds: 1 (2.3 mg, R_f = 0.52), 2 (4.5 mg, R_f = 0.65), 3 (2.03 g, R_f = 0.14), 4 (20.0 mg, R_f = 0.40), 5 (5.0 mg, R_f = 0.60), 6 (3.4 mg, R_f = 0.16), 7 (2.0 mg, R_f = 0.09), 8 (10.2 mg, R_f = 0.56), 9 (6.5 mg, R_f = 0.62), 10 (10.3 mg, R_f = 0.44), 11 (100 mg, R_f = 0.46), 12 (20.6 mg, R_f = 0.20), and 13 (6.0 mg, R_f = 0.43).

Fr.3 (6.0 g) was subjected to column chromatography (3 × 35 cm) using a MeOH/CHCl₃ (0 - 30 %) gradient system to afford five subfractions 1 - 5. Subfraction 3 - 2 yielded 3 (0.5 g), 12 (1.12 g), and 14 (5.0 mg, R_f = 0.31) by reversed-phase preparative TLC with MeCN/acetone/H₂O (1 : 1:1).

Fr.4 (5.0 g) was also subjected to silica gel column chromatography (3 × 35 cm) with EtOAc/hexane and then MeOH/CHCl₃ to give five subfractions 1 - 5. Purification of fr.4 - 4 by reversed-phase preparative TLC with MeCN/acetone/H₂O (1 : 1:1) yielded 3 (30.5), 12 (50.4 mg), and 15 (10.0 mg, R_f = 0.36).

7-Oxo-13α-hydroxyabiet-8(14)-en-18-oic acid (1): Colorless amorphous solid; [α]_D ²²: -27° (c 0.14 CHCl₃); IR (CHCl₃): ν_max = 3600, 1690, 1460, 1390 cm^-1; ¹H- and ¹³C-NMR see Table [1]; HR-EI-MS: m/z = [M⁺] 334.2148 (calcd for C₂₀H₃₀O₄: 334.2144).

13,14-Seco-13-oxoabiet-7-en-14,18-dioic acid (2): Colorless amorphous solid; [α]_D ²⁴: -6° (c 0.3 CHCl₃); IR (CHCl₃): ν_max = 1710, 1700, 1690, 1460, 1270 cm^-1; ¹H- and ¹³C-NMR see Table [1]; HR-EI-MS: m/z = [M⁺] 350.2110 (calcd for C₂₀H₃₀O₅: 350.2093).

PANC-1 preferential cytotoxicity assay was done as described earlier [3] and is provided in the Supporting Information. Briefly, PANC-1 cancer cells were seeded in 96-well plates (1 × 10⁴/well) and incubated in fresh Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceuticals; Tokyo, Japan) at 37 °C under 5 % CO₂ and 95 % air for 24 h. The nutrient-deprived medium (NDM) was prepared following the procedure described by Esumi et al [3]. After the cells were washed with PBS (Nissui Pharmaceuticals), the medium was changed to either DMEM or NDM and serial dilutions of the test samples were added. After 24 h incubation, the cells were washed with PBS, and 100 μL of DMEM containing 10 % WST-8 cell counting kit (Dojindo; Kumamoto, Japan) solution was added to the wells. After 2 h incubation, absorbance at 450 nm was measured. Cell viability was calculated from the mean values of data from three wells.

Cell viability (%) =
[{Abs_{(test sample)} - Abs_(blank)}/{Abs_(control) - Abs_(blank)}] × 100

Acknowledgements

This work was supported in part by a grant from the Ministry of Health and Welfare for the Second-Term Comprehensive 10-year Strategy for Cancer Control, Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare and grant from the Ministry of Education, Culture, Sports, Science and Technology.

References

• 1 Li D, Xie K, Wolff R, Abbruzzese J L. Pancreatic cancer.  Lancet. 2004;  363 1049-57
  CrossrefPubMedSearch in Google Scholar
• 2 Shore S, Vimalachandran D, Raraty M GT, Ghaneh P. Cancer in the elderly: pancreatic cancer.  Surg Oncol. 2004;  13 201-10
  CrossrefPubMedSearch in Google Scholar
• 3 Izushi K, Kato K, Ogura T, Kinoshita T, Esumi H. Remarkable tolerance of tumor cells to nutrient deprivation: possible new biochemical target for cancer therapy.  Cancer Res. 2000;  60 6201-7
  PubMedSearch in Google Scholar
• 4 Awale S, Lu J, Kalauni S K, Kurashima Y, Tezuka Y, Kadota S. et al . Identification of arctigenin as an antitumor agent having the ability to eliminate the tolerance of cancer cells to nutrient starvation.  Cancer Res. 2006;  66 1751-7
  CrossrefPubMedSearch in Google Scholar
• 5 Awale S, Nakashima E MN, Kalauni S K, Tezuka Y, Kurashima Y, Lu J. et al . Angelmarin, a novel anti-cancer agent able to eliminate the tolerance of cancer cells to nutrient starvation.  Bioorg Med Chem Lett. 2006;  16 581-3
  CrossrefPubMedSearch in Google Scholar
• 6 Lu J, Kunimoto S, Yamazaki Y, Kaminishi M, Esumi H. Kigamicin D, a novel anticancer agent based on a new anti-austerity strategy targeting cancer cells’ tolerance to nutrient starvation.  Cancer Sci. 2004;  95 547-52
  CrossrefPubMedSearch in Google Scholar
• 7 Zinkel D, Russel J. Naval stores: production, chemistry, utilization. In: Zinkel D, Russel J, editors Atlanta; Pine Chemicals Association 1989
  Search in Google Scholar
• 8 Usmanghani K, Saeed A, Alam M T. Indusynic Medicine. In: Usmanghani K, editor 1st edition A-952. Karachi; Research Institute of Indusyunic Medicine 1997: p 338-9
  Search in Google Scholar
• 9 William M C. Non-wood forest products from conifers. Rome; FAO-Food and Agriculture Organization of the United Nations 1995: p 1-15
  Search in Google Scholar
• 10 Namba T. Coloured illustrations of Wakan-Yaku (The crude drugs in Japan, China and the neighbouring countries). In: Namba T, editor Vol II Osaka; Hoikusha Publishing Co, Ltd 1980: p 24-5
  Search in Google Scholar
• 11 Ruzicka L, Sternbach L. Zur Kenntnis der Diterpene.  Helv Chim Acta. 1940;  23 341-55
  CrossrefPubMedSearch in Google Scholar
• 12 Darling T R, Turro N J. Carbon-13 nuclear magnetic resonances spectroscopy of naturally occurring substances. X. Pimaradienes.  J Am Chem Soc. 1972;  94 4367-9
  CrossrefPubMedSearch in Google Scholar
• 13 Ayer W A, Macaulay J B. Metabolites of the honey mushroom, Armillaria mellea .  Can J Chem. 1987;  65 7-13
  CrossrefPubMedSearch in Google Scholar
• 14 Bol’shakova V I, Shmidt E N, Pentegova V A, Mamatyuk V I. Minor constituents of the resin of the Kurile Dahurian, Japanese and Siberian larches.  Khim Prir Soedin. 1986;  5 571-6
  PubMedSearch in Google Scholar
• 15 Erdtman H, Malmborg L. The Beckmann rearrangement of the oxime of 7-ketodehydroabietate. A side reaction.  Acta Chem Scand. 1970;  24 2252
  PubMedSearch in Google Scholar
• 16 Chamy M C, Piovano M, Gambaro V, Garbarino J A, Nicoletti M. Dehydroabietane diterpenoids from Calceolaria ascendens .  Phytochemistry. 1987;  26 1763-5
  CrossrefPubMedSearch in Google Scholar
• 17 Cheung H TA, Miyase T, Lenguyen M P, Smal M A. Further acidic and neutral components of Pinus massoniana Resin.  Tetrahedron. 1993;  49 7903-15
  CrossrefPubMedSearch in Google Scholar
• 18 Kinouchi Y, Ohtsu H, Tokuda H, Nishino H, Matsunaga S, Tanaka R. Potential antitumor-promoting diterpenoids from the stem bark of Picea glehni .  J Nat Prod. 2000;  63 817-20
  CrossrefPubMedSearch in Google Scholar
• 19 Ohtsu H, Tanaka R, In Y, Matsunaga S, Tokuda H, Nishino H. Abietane diterpenoids from the cones of Larix kaempferi and their inhibitory effects on Epstein-Barr virus activation.  Planta Med. 2001;  67 55-60
  Thieme ConnectPubMedSearch in Google Scholar
• 20 Tanaka R, Ohtsu H, Matsunaga S. Abietane diterpene acids and other constituents from the leaves of Larix kaempferi .  Phytochemistry. 1997;  46 1051-7
  CrossrefPubMedSearch in Google Scholar
• 21 Roshchin V I, Kolodynskaya L A, Raldugin V A. Abietic and dehydroabietic acid derivatives from the needle-free shoots of Pinus sylvestris .  Khim Prir Soedin. 1985;  3 345-51
  PubMedSearch in Google Scholar

Dr. Prof. Shigetoshi Kadota

Institute of Natural Medicine

University of Toyama

2630 Sugitani

Toyama 930-0194

Japan

Phone: +81-76-434-7625

Fax: +81-76-434-5059

Email: kadota@ms.toyama-mpu.ac.jp

References

• 1 Li D, Xie K, Wolff R, Abbruzzese J L. Pancreatic cancer.  Lancet. 2004;  363 1049-57
  CrossrefPubMedSearch in Google Scholar
• 2 Shore S, Vimalachandran D, Raraty M GT, Ghaneh P. Cancer in the elderly: pancreatic cancer.  Surg Oncol. 2004;  13 201-10
  CrossrefPubMedSearch in Google Scholar
• 3 Izushi K, Kato K, Ogura T, Kinoshita T, Esumi H. Remarkable tolerance of tumor cells to nutrient deprivation: possible new biochemical target for cancer therapy.  Cancer Res. 2000;  60 6201-7
  PubMedSearch in Google Scholar
• 4 Awale S, Lu J, Kalauni S K, Kurashima Y, Tezuka Y, Kadota S. et al . Identification of arctigenin as an antitumor agent having the ability to eliminate the tolerance of cancer cells to nutrient starvation.  Cancer Res. 2006;  66 1751-7
  CrossrefPubMedSearch in Google Scholar
• 5 Awale S, Nakashima E MN, Kalauni S K, Tezuka Y, Kurashima Y, Lu J. et al . Angelmarin, a novel anti-cancer agent able to eliminate the tolerance of cancer cells to nutrient starvation.  Bioorg Med Chem Lett. 2006;  16 581-3
  CrossrefPubMedSearch in Google Scholar
• 6 Lu J, Kunimoto S, Yamazaki Y, Kaminishi M, Esumi H. Kigamicin D, a novel anticancer agent based on a new anti-austerity strategy targeting cancer cells’ tolerance to nutrient starvation.  Cancer Sci. 2004;  95 547-52
  CrossrefPubMedSearch in Google Scholar
• 7 Zinkel D, Russel J. Naval stores: production, chemistry, utilization. In: Zinkel D, Russel J, editors Atlanta; Pine Chemicals Association 1989
  Search in Google Scholar
• 8 Usmanghani K, Saeed A, Alam M T. Indusynic Medicine. In: Usmanghani K, editor 1st edition A-952. Karachi; Research Institute of Indusyunic Medicine 1997: p 338-9
  Search in Google Scholar
• 9 William M C. Non-wood forest products from conifers. Rome; FAO-Food and Agriculture Organization of the United Nations 1995: p 1-15
  Search in Google Scholar
• 10 Namba T. Coloured illustrations of Wakan-Yaku (The crude drugs in Japan, China and the neighbouring countries). In: Namba T, editor Vol II Osaka; Hoikusha Publishing Co, Ltd 1980: p 24-5
  Search in Google Scholar
• 11 Ruzicka L, Sternbach L. Zur Kenntnis der Diterpene.  Helv Chim Acta. 1940;  23 341-55
  CrossrefPubMedSearch in Google Scholar
• 12 Darling T R, Turro N J. Carbon-13 nuclear magnetic resonances spectroscopy of naturally occurring substances. X. Pimaradienes.  J Am Chem Soc. 1972;  94 4367-9
  CrossrefPubMedSearch in Google Scholar
• 13 Ayer W A, Macaulay J B. Metabolites of the honey mushroom, Armillaria mellea .  Can J Chem. 1987;  65 7-13
  CrossrefPubMedSearch in Google Scholar
• 14 Bol’shakova V I, Shmidt E N, Pentegova V A, Mamatyuk V I. Minor constituents of the resin of the Kurile Dahurian, Japanese and Siberian larches.  Khim Prir Soedin. 1986;  5 571-6
  PubMedSearch in Google Scholar
• 15 Erdtman H, Malmborg L. The Beckmann rearrangement of the oxime of 7-ketodehydroabietate. A side reaction.  Acta Chem Scand. 1970;  24 2252
  PubMedSearch in Google Scholar
• 16 Chamy M C, Piovano M, Gambaro V, Garbarino J A, Nicoletti M. Dehydroabietane diterpenoids from Calceolaria ascendens .  Phytochemistry. 1987;  26 1763-5
  CrossrefPubMedSearch in Google Scholar
• 17 Cheung H TA, Miyase T, Lenguyen M P, Smal M A. Further acidic and neutral components of Pinus massoniana Resin.  Tetrahedron. 1993;  49 7903-15
  CrossrefPubMedSearch in Google Scholar
• 18 Kinouchi Y, Ohtsu H, Tokuda H, Nishino H, Matsunaga S, Tanaka R. Potential antitumor-promoting diterpenoids from the stem bark of Picea glehni .  J Nat Prod. 2000;  63 817-20
  CrossrefPubMedSearch in Google Scholar
• 19 Ohtsu H, Tanaka R, In Y, Matsunaga S, Tokuda H, Nishino H. Abietane diterpenoids from the cones of Larix kaempferi and their inhibitory effects on Epstein-Barr virus activation.  Planta Med. 2001;  67 55-60
  Thieme ConnectPubMedSearch in Google Scholar
• 20 Tanaka R, Ohtsu H, Matsunaga S. Abietane diterpene acids and other constituents from the leaves of Larix kaempferi .  Phytochemistry. 1997;  46 1051-7
  CrossrefPubMedSearch in Google Scholar
• 21 Roshchin V I, Kolodynskaya L A, Raldugin V A. Abietic and dehydroabietic acid derivatives from the needle-free shoots of Pinus sylvestris .  Khim Prir Soedin. 1985;  3 345-51
  PubMedSearch in Google Scholar

Dr. Prof. Shigetoshi Kadota

Institute of Natural Medicine

University of Toyama

2630 Sugitani

Toyama 930-0194

Japan

Phone: +81-76-434-7625

Fax: +81-76-434-5059

Email: kadota@ms.toyama-mpu.ac.jp

• Supplementary Material